T1R taste receptors and genes encoding same

ABSTRACT

Binding assays for identifying components that modulate human T1R1 polypeptide-associated taste are disclosed. These assays detect the specific binding of compounds to a human T1R1 polypeptide or the modulation of the specific binding of another compound that specifically binds human T1R1 polypeptide. The binding assays may include the use of detectable labels, e.g., radionuclides, enxymes, fluorophores, and the like. Compounds identified in these binding assays have potential application as T1R1 taste modulators and can be used as additives in compositions for human or animal consumption.

RELATED APPLICATIONS

[0001] This application is related to the following provisionalapplications: U.S. Ser. No. 60/187,546, filed Mar. 7, 2000, entitled,“NOVEL TASTE RECEPTOR AND GENE ENCODING SAME,” to Zozulya and Adler;U.S. Ser. No. 60/195,536, filed Apr. 7, 2000, entitled, “MAMMALIAN TASTERECEPTOR AND HUMAN ORTHOLOG,” to Adler; U.S. Ser. No. 60/209,840, filedJun. 6, 2000, entitled, “NOVEL TASTE RECEPTOR AND GENE ENCODING SAME,”to Zozulya and Adler, U.S. Ser. No. 60/214,213, filed Jun. 23, 2000,entitled, “NOVEL TASTE RECEPTOR AND GENE ENCODING SAME,” to Zozulya andAdler; U.S. Ser. No. 60/226,448, filed Aug. 17, 2000, entitled, “NOVELTASTE RECEPTOR AND GENE ENCODING SAME,” to Zozulya and Adler, and U.S.Ser. No. 60/259,227, filed Jan. 3, 2001, entitled “T1R TASTE RECEPTORSAND GENES ENCODING SAME,” to Adler, Li, Staszewski, and O'Connell, whichare all herein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to newly identified mammalian chemosensoryG protein-coupled receptors, to family of such receptors, and to thegenes and cDNA encoding said receptors. More particularly, the inventionrelates to newly identified mammalian chemosensory G protein-coupledreceptors active in taste signaling, to a family of such receptors, tothe genes and cDNA encoding said receptors, and to methods of using suchreceptors, genes, and cDNA in the analysis and discovery of tastemodulators.

[0004] 2. Description of the Related Art

[0005] The taste system provides sensory information about the chemicalcomposition of the external world. Taste transduction is one of the mostsophisticated forms of chemical-triggered sensation in animals. Atpresent, the means by which taste sensations are elicited remains poorlyunderstood. See, e.g., Margolskee, BioEssays, 15:645-50(1993); Avenet etal., J. Membrane Biol., 112:1-8(1989). Taste signaling is foundthroughout the animal kingdom, from simple metazoans to the most complexof vertebrates. Taste sensation is thought to involve distinct signalingpathways. These pathways are believed to be mediated by receptors, i.e.,metabotropic or inotropic receptors. Cells which express tastereceptors, when exposed to certain chemical stimuli, elicit tastesensation by depolarizing to generate an action potential, which isbelieved to trigger the sensation. This event is believed to trigger therelease of neurotransmitters at gustatory afferent neuron synapses,thereby initiating signaling along neuronal pathways that mediate tasteperception. See, e.g., Roper, Ann. Rev. Neurosci., 12:329-53 (1989).

[0006] As such, taste receptors specifically recognize molecules thatelicit specific taste sensation. These molecules are also referred toherein as “tastants.” Many taste receptors belong to the 7-transmembranereceptor superfamily (Hoon et al., Cell 96:451 (1999); Adler et al, Cell100:693 (2000)), which are also known as G protein-coupled receptors(GPCRs). Other tastes are believed to be mediated by channel proteins. Gprotein-coupled receptors control many physiological functions, such asendocrine function, exocrine function, heart rate, lipolysis,carbohydrate metabolism, and transmembrane signaling. The biochemicalanalysis and molecular cloning of a number of such receptors hasrevealed many basic principles regarding the function of thesereceptors.

[0007] For example, U.S. Pat. No. 5,691,188 describes how upon a ligandbinding to a GPCR, the receptor presumably undergoes a conformationalchange leading to activation of the G protein. G proteins are comprisedof three subunits: a guanyl nucleotide binding α subunit, a β subunit,and a γ subunit. G proteins cycle between two forms, depending onwhether GDP or GTP is bound to the α subunit. When GDP is bound, the Gprotein exists as a heterotrimer: the Gαβγ complex. When GTP is bound,the α subunit dissociates from the heterotrimer, leaving a Gβγ complex.When a Gαβγ complex operatively associates with an activated Gprotein-coupled receptor in a cell membrane, the rate of exchange of GTPfor bound GDP is increased and the rate of dissociation of the bound Gαsubunit from the Gαβγ complex increases. The free Gα subunit and Gβγcomplex are thus capable of transmitting a signal to downstream elementsof a variety of signal transduction pathways. These events form thebasis for a multiplicity of different cell signaling phenomena,including for example the signaling phenomena that are identified asneurological sensory perceptions such as taste and/or smell.

[0008] Mammals are believed to have five basic taste modalities: sweet,bitter, sour, salty, and umami (the taste of monosodium glutamate). See,e.g., Kawamura et al., Introduction to Umami: A Basic Taste (1987);Kinnamon et al., Ann. Rev. Physiol., 54:715-31 (1992); Lindemann,Physiol. Rev., 76:718-66 (1996); Stewart et al., Am. J. Physiol.,272:1-26(1997). Numerous physiological studies in animals have shownthat taste receptor cells may selectively respond to different chemicalstimuli. See, e.g., Akabas et al., Science, 242:1047-50 (1988);Gilbertson et al., J. Gen. Physiol., 100:803-24 (1992); Bernhardt etal., J. Physiol., 490:325-36 (1996); Cummings et al., J. Neurophysiol.,75:1256-63 (1996).

[0009] In mammals, taste receptor cells are assembled into taste budsthat are distributed into different papillae in the tongue epithelium.Circumvallate papillae, found at the very back of the tongue, containhundreds to thousands of taste buds. By contrast, foliate papillae,localized to the posterior lateral edge of the tongue, contain dozens tohundreds of taste buds. Further, fungiform papillae, located at thefront of the tongue, contain only a single or a few taste buds.

[0010] Each taste bud, depending on the species, contains 50-150 cells,including precursor cells, support cells, and taste receptor cells. See,e.g., Lindemann, Physiol. Rev., 76:718-66 (1996). Receptor cells areinnervated at their base by afferent nerve endings that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. Elucidating the mechanisms of taste cellsignaling and information processing is important to understanding thefunction, regulation, and perception of the sense of taste.

[0011] Although much is known about the psychophysics and physiology oftaste cell function, very little is known about the molecules andpathways that mediate its sensory signaling response. The identificationand isolation of novel taste receptors and taste signaling moleculescould allow for new methods of chemical and genetic modulation of tastetransduction pathways. For example, the availability of receptor andchannel molecules could permit the screening for high affinity agonists,antagonists, inverse agonists, and modulators of taste activity. Suchtaste modulating compounds could be useful in the pharmaceutical andfood industries to improve the taste of a variety of consumer products,or to block undesirable tastes, e.g., in certain pharmaceuticals.

[0012] Complete or partial sequences of numerous human and othereukaryotic chemosensory receptors are currently known. See, e.g.,Pilpel, Y. and Lancet, D., Protein Science, 8:969-977 (1999); Mombaerts,P., Annu. Rev. Neurosci., 22:487-50 (1999). See also, EP0867508A2, U.S.Pat. No. 5,874,243, WO 92/17585, WO 95/18140, WO 97/17444, WO 99/67282.Because of the complexity of ligand-receptor interactions, and moreparticularly tastant-receptor interactions, information aboutligand-receptor recognition is lacking. In part, the present inventionaddresses the need for better understanding of the interactions betweenchemosensory receptors and chemical stimuli. The present invention alsoprovides, among other things, novel chemosensory receptors, and methodsfor utilizing such receptors, and the genes a cDNAs encoding suchreceptors, to identify molecules that can be used to modulatechemosensory transduction, such as taste sensation.

SUMMARY OF THE INVENTION

[0013] The invention relates to a new family of G protein-coupledreceptors, and to the genes and cDNAs encoding said receptors. Thereceptors are thought to be primarily involved in sweet tastetransduction, but can be involved in transducing signals from othertaste modalities as well.

[0014] The invention provides methods for representing the perception oftaste and/or for predicting the perception of taste in a mammal,including in a human. Preferably, such methods may be performed by usingthe receptors and genes encoding said receptors disclosed herein.

[0015] Toward that end, it is an object of the invention to provide anew family of mammalian G protein-coupled receptors, herein referred toas T1Rs, active in taste perception. It is another object of theinvention to provide fragments and variants of such T1Rs that retaintastant-binding activity.

[0016] It is yet another object of the invention to provide nucleic acidsequences or molecules that encode such T1Rs, fragments, or variantsthereof.

[0017] It is still another object of the invention to provide expressionvectors which include nucleic acid sequences that encode such T1Rs, orfragments or variants thereof, which are operably linked to at least oneregulatory sequence such as a promoter, enhancer, or other sequenceinvolved in positive or negative gene transcription and/or translation.

[0018] It is still another object of the invention to provide human ornon-human cells that functionally express at least one of such T1Rs, orfragments or variants thereof.

[0019] It is still another object of the invention to provide T1R fusionproteins or polypeptides which include at least a fragment of at leastone of such T1Rs.

[0020] It is another object of the invention to provide an isolatednucleic acid molecule encoding a T1R polypeptide comprising a nucleicacid sequence that is at least 50%, preferably 75%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, andconservatively modified variants thereof.

[0021] It is a further object of the invention to provide an isolatednucleic acid molecule comprising a nucleic acid sequence that encodes apolypeptide having an amino acid sequence at least 35 to 50%, andpreferably 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toan amino acid sequence selected from the group consisting of: SEQ IDNOS: 4, 10, 12, 14, 17, and conservatively modified variants thereof,wherein the fragment is at least 20, preferably 40, 60, 80, 100, 150,200, or 250 amino acids in length. Optionally, the fragment can be anantigenic fragment which binds to an anti-T1R antibody.

[0022] It is still a further object of the invention to provide anisolated polypeptide comprising a variant of said fragment, whereinthere is a variation in at most 10, preferably 5, 4, 3, 2, or 1 aminoacid residues.

[0023] It is still another object of the invention to provide agonistsor antagonists of such T1Rs, or fragments or variants thereof.

[0024] It is yet another object of the invention to provide methods forrepresenting the perception of taste and/or for predicting theperception of taste in a mammal, including in a human. Preferably, suchmethods may be performed by using the T1Rs, or fragments or variantsthereof, and genes encoding such T1Rs, or fragments or variants thereof,disclosed herein.

[0025] It is yet another object of the invention to provide novelmolecules or combinations of molecules which elicit a predeterminedtaste perception in a mammal. Such molecules or compositions can begenerated by determining a value of taste perception in a mammal for aknown molecule or combinations of molecules; determining a value oftaste perception in a mammal for one or more unknown molecules orcombinations of molecules; comparing the value of taste perception in amammal for one or more unknown compositions to the value of tasteperception in a mammal for one or more known compositions; selecting amolecule or combination of molecules that elicits a predetermined tasteperception in a mammal; and combining two or more unknown molecules orcombinations of molecules to form a molecule or combination of moleculesthat elicits a predetermined taste perception in a mammal. The combiningstep yields a single molecule or a combination of molecules that elicitsa predetermined taste perception in a mammal.

[0026] It is still a further object of the invention to provide a methodof screening one or more compounds for the presence of a tastedetectable by a mammal, comprising: a step of contacting said one ormore compounds with at least one of the disclosed T1Rs, fragments orvariants thereof, preferably wherein the mammal is a human.

[0027] It is another object of the invention to provided a method forsimulating a taste, comprising the steps of: for each of a plurality ofT1Rs, or fragments of variants thereof disclosed herein, preferablyhuman T1Rs, ascertaining the extent to which the T1R interacts with thetastant; and combining a plurality of compounds, each having apreviously ascertained interaction with one or more of the T1Rs, inamounts that together provide a receptor-stimulation profile that mimicsthe profile for the taste. Interaction of a tastant with a T1R can bedetermined using any of the binding or reporter assays described herein.The plurality of compounds may then be combined to form a mixture. Ifdesired, one or more of the plurality of the compounds can be combinedcovalently. The combined compounds substantially stimulate at least 50%,60%, 70%, 75%, 80% or 90% or all of the receptors that are substantiallystimulated by the tastant.

[0028] In yet another aspect of the invention, a method is providedwherein a plurality of standard compounds are tested against a pluralityof T1Rs, or fragments or variants thereof, to ascertain the extent towhich the T1Rs each interact with each standard compound, therebygenerating a receptor stimulation profile for each standard compound.These receptor stimulation profiles may then be stored in a relationaldatabase on a data storage medium. The method may further compriseproviding a desired receptor-stimulation profile for a taste; comparingthe desired receptor stimulation profile to the relational database; andascertaining one or more combinations of standard compounds that mostclosely match the desired receptor-stimulation profile. The method mayfurther comprise combining standard compounds in one or more of theascertained combinations to simulate the taste.

[0029] It is a further object of the invention to provide a method forrepresenting taste perception of a particular tastant in a mammal,comprising the steps of: providing values X₁ to X_(n) representative ofthe quantitative stimulation of each of n T1Rs of said vertebrate, wheren is greater than or equal to 2; and generating from said values aquantitative representation of taste perception. The T1Rs may be antaste receptor disclosed herein, or fragments or variants thereof, therepresentation may constitutes a point or a volume in n-dimensionalspace, may constitutes a graph or a spectrum, and may constitutes amatrix of quantitative representations. Also, the providing step maycomprise contacting a plurality of recombinantly-produced T1Rs, orfragments or variants thereof, with a test composition andquantitatively measuring the interaction of said composition with saidreceptors.

[0030] It is yet another object of the invention to provide a method forpredicting the taste perception in a mammal generated by one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal, comprising the steps of: providing values X₁ to X_(n)representative of the quantitative stimulation of each of n T1Rs of saidvertebrate, where n is greater than or equal to 2; for one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal, providing values X₁ to X_(n) representative of thequantitative stimulation of each of n T1Rs of said vertebrate, where nis greater than or equal to 2; for one or more molecules or combinationsof molecules yielding unknown taste perception in a mammal; andgenerating from said values a quantitative representation of tasteperception in a mammal for the one or more molecules or combinations ofmolecules yielding unknown taste perception in a mammal, and predictingthe taste perception in a mammal generated by one or more molecules orcombinations of molecules yielding unknown taste perception in a mammalby comparing the quantitative representation of taste perception in amammal for the one or more molecules or combinations of moleculesyielding unknown taste perception in a mammal to the quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal. The T1Rs used in this method may include a taste receptor,or fragment or variant thereof, disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a cryosection of frozen mouse tongue showing T1R3 geneexpression in taste buds of mouse circumvallate papillae by in situhybridization. Select T1R3-expressing taste receptor cells are markedwith arrows.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention thus provides isolated nucleic acid moleculesencoding taste-cell-specific G protein-coupled receptors (“GPCR”), andthe polypeptides they encode. These nucleic acid molecules and thepolypeptides that they encode are members of the T1R family oftaste-cell-specific GPCRs. Members of the T1R family oftaste-cell-specific GPCRs are identified in Hoon et al., Cell96:541-551(1999), WO 00/06592, and WO 00/06593, all of which are incorporatedherein by reference in their entireties.

[0033] More particularly, the invention provides nucleic acids encodinga novel family of taste-cell-specific GPCRs. These nucleic acids and thereceptors that they encode are referred to as members of the “T1R”family of taste-cell-specific GPCRs. In particular embodiments of theinvention, the T1R family members include rT1R3, mT1R3, hT1R3, andhT1R1. While not wishing to be bound by theory, it is believed thatthese taste-cell-specific GPCRs are components of the taste transductionpathway, and may be involved in the taste detection of sweet substancesand/or other taste modalities.

[0034] Further, it is believed that T1R family members may act incombination with other T1R family members, other taste-cell-specificGPCRs, or a combination thereof, to thereby effect chemosensory tastetransduction. For instance, it is believed that T1R1 and T1R3 maybecoexressed within the same taste receptor cell type, and the tworeceptors may physically interact to form a heterodimeric tastereceptor. Alternatively, T1R1 and T1R3 may both independently bind tothe same type of ligand, and their combined binding may result in aspecific perceived taste sensation.

[0035] These nucleic acids provide valuable probes for theidentification of taste cells, as the nucleic acids are specificallyexpressed in taste cells. For example, probes for T1R polypeptides andproteins can be used to identify taste cells present in foliate,circumvallate, and fungiform papillae, as well as taste cells present inthe geschmackstreifen, oral cavity, gastrointestinal epithelium, andepiglottis. They may also serve as tools for the generation of tastetopographic maps that elucidate the relationship between the taste cellsof the tongue and taste sensory neurons leading to taste centers in thebrain. In particular, methods of detecting T1Rs can be used to identifytaste cells sensitive to sweet tastants or other specific modalities oftastants. Furthermore, the nucleic acids and the proteins they encodecan be used as probes to dissect taste-induced behaviors. Also,chromosome localization of the genes encoding human T1Rs can be used toidentify diseases, mutations, and traits caused by and associated withT1R family members.

[0036] The nucleic acids encoding the T1R proteins and polypeptides ofthe invention can be isolated from a variety of sources, geneticallyengineered, amplified, synthesized, and/or expressed recombinantlyaccording to the methods disclosed in WO 00/035374, which is hereinincorporated by reference in its entirety.

[0037] The invention also provides methods of screening for modulators,e.g., activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel taste-cell-specific GPCRs. Such modulatorsof taste transduction are useful for pharmacological, chemical, andgenetic modulation of taste signaling pathways. These methods ofscreening can be used to identify high affinity agonists and antagonistsof taste cell activity. These modulatory compounds can then be used inthe food and pharmaceutical industries to customize taste, e.g., tomodulate the sweet tastes of foods or drugs.

[0038] Thus, the invention provides assays for detecting andcharacterizing taste modulation, wherein T1R family members act asdirect or indirect reporter molecules of the effect of modulators ontaste transduction. GPCRs can be used in assays to, e.g., measurechanges in ligand binding, ion concentration, membrane potential,current flow, ion flux, transcription, signal transduction,receptor-ligand interactions, second messenger concentrations, in vitro,in vivo, and ex vivo. In one embodiment, members of the T1R family canbe used as indirect reporters via attachment to a second reportermolecule such as green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology, 15:961-964 (1997)). In anotherembodiment, T1R family members may be recombinantly expressed in cells,and modulation of taste transduction via GPCR activity may be assayed bymeasuring changes in Ca²⁺ levels and other intracellular messages suchas cAMP, cGMP, or IP3.

[0039] In certain embodiments, a domain of a T1R polypeptide, e.g., anextracellular, transmembrane, or intracellular domain, is fused to aheterologous polypeptide, thereby forming a chimeric polypeptide, e.g.,a chimeric polypeptide with GPCR activity. Such chimeric polypeptidesare useful, e.g., in assays to identify ligands, agonists, antagonists,or other modulators of a T1R polypeptide. In addition, such chimericpolypeptides are useful to create novel taste receptors with novelligand binding specificity, modes of regulation, signal transductionpathways, or other such properties, or to create novel taste receptorswith novel combinations of ligand binding specificity, modes ofregulation, signal transduction pathways, etc.

[0040] In one embodiment, a T1R polypeptide is expressed in a eukaryoticcell as a chimeric receptor with a heterologous, chaperone sequence thatfacilitates plasma membrane trafficking, or maturation and targetingthrough the secretory pathway. The optional heterologous sequence may bea rhodopsin sequence, such as an N-terminal fragment of a rhodopsin.Such chimeric T1R receptors can be expressed in any eukaryotic cell,such as HEK-293 cells. Preferably, the cells comprise a G protein, e.g.,Gα15 or Gα16 or another type of promiscuous G protein capable of pairinga wide range of chemosensory GPCRs to an intracellular signaling pathwayor to a signaling protein such as phospholipase C. Activation of suchchimeric receptors in such cells can be detected using any standardmethod, such as by detecting changes in intracellular calcium bydetecting FURA-2 dependent fluorescence in the cell. If preferred hostcells do not express an appropriate G protein, they may be transfectedwith a gene encoding a promiscuous G protein such as those described inU.S. application Ser. No. 60/243,770, which is herein incorporated byreference in its entirety.

[0041] Methods of assaying for modulators of taste transduction includein vitro ligand-binding assays using: T1R polypeptides, portionsthereof, i.e., the extracellular domain, transmembrane region, orcombinations thereof, or chimeric proteins comprising one or moredomains of a T1R family member; oocyte or tissue culture cellsexpressing T1R polypeptides, fragments, or fusion proteins;phosphorylation and dephosphorylation of T1R family members; G proteinbinding to GPCRs; ligand-binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cGMP, CAMP and inositol triphosphate; changes inintracellular calcium levels; and neurotransmitter release.

[0042] Further, the invention provides methods of detecting T1R nucleicacid and protein expression, allowing investigation of tastetransduction regulation and specific identification of taste receptorcells. T1R family members also provide useful nucleic acid probes forpaternity and forensic investigations. T1R genes are also useful as anucleic acid probes for identifying taste receptor cells, such asfoliate, fungiform, circumvallate, geschmackstreifen, and epiglottistaste receptor cells. T1R receptors can also be used to generatemonoclonal and polyclonal antibodies useful for identifying tastereceptor cells. Taste receptor cells can be identified using techniquessuch as reverse transcription and amplification of RNA, isolation oftotal RNA or poly A+ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, S1 digestion, probing DNA microchiparrays, western blots, and the like.

[0043] Functionally, the T1R polypeptides comprise a family of relatedseven transmembrane G protein-coupled receptors, which are believed tobe involved in taste transduction and may interact with a G protein tomediate taste signal transduction (see, e.g., Fong, Cell Signal, 8:217(1996); Baldwin, Curr. Opin. Cell Biol., 6:180 (1994)). Structurally,the nucleotide sequences of T1R family members may encode relatedpolypeptides comprising an extracellular domain, seven transmembranedomains, and a cytoplasmic domain. Related T1R family genes from otherspecies share at least about 50%, and optionally 60%, 70%, 80%, or 90%,nucleotide sequence identity over a region of at least about 50nucleotides in length, optionally 100, 200, 500, or more nucleotides inlength to SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, or conservativelymodified variants thereof, or encode polypeptides sharing at least about35 to 50%, and optionally 60%, 70%, 80%, or 90%, amino acid sequenceidentity over an amino acid region at least about 25 amino acids inlength, optionally 50 to 100 amino acids in length to SEQ ID NOS: 4,,10, 12, 14, 17, or conservatively modified variants thereof.

[0044] Several consensus amino acid sequences or domains have also beenidentified that are characteristic of T1R family members. For example,T1R family members typically comprise a sequence having at least about50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95-99%, orhigher, identity to T1R consensus sequences 1 and 2 (SEQ ID NOs 18 and19, respectively). These conserved domains thus can be used to identifymembers of the T1R family, by identity, specific hybridization oramplification, or specific binding by antibodies raised against adomain. Such T1R consensus sequences have the following amino acidsequences:

[0045] T1R Family Consensus Sequence 1: (SEQ ID NO: 18)

[0046] (TR)C(FL)(RQP)R(RT)(SPV)(VERKT)FL(AE)(WL)(RHG)E

[0047] T1R Family Consensus Sequence 2: (SEQ ID NO: 19)

[0048] (LQ)P(EGT)(NRC)YN(RE)A(RK)(CGF)(VLI)T(FL)(AS)(ML)

[0049] These consensus sequences are inclusive of those found in the T1Rpolypeptides described herein, but T1R family members from otherorganisms may be expected to comprise consensus sequences having about75% identity or more to the inclusive consensus sequences describedspecifically herein.

[0050] Specific regions of the T1R nucleotide and amino acid sequencesmay be used to identify polymorphic variants, interspecies homologs, andalleles of T1R family members. This identification can be made in vitro,e.g., under stringent hybridization conditions or PCR (e.g., usingprimers encoding the T1R consensus sequences identified above), or byusing the sequence information in a computer system for comparison withother nucleotide sequences. Different alleles of T1R genes within asingle species population will also be useful in determining whetherdifferences in allelic sequences correlate to differences in tasteperception between members of the population. Classical PCR-typeamplification and cloning techniques are useful for isolating orthologs,for example, where degenerate primers are sufficient for detectingrelated genes across species, which typically have a higher level ofrelative identity than paralogous members of the T1R family within asingle species.

[0051] For instance, degenerate primers SAP077 (SEQ. ID NO. 5) andSAP0079 (SEQ. ID NO. 6) can be used can be used to amplify and cloneT1R3 genes from different mammalian genomes. In contrast, genes within asingle species that are related to T1R3 are best identified usingsequence pattern recognition software to look for related sequences.Typically, identification of polymorphic variants and alleles of T1Rfamily members can be made by comparing an amino acid sequence of about25 amino acids or more, e.g., 50-100 amino acids. Amino acid identity ofapproximately at least 35 to 50%, and optionally 60%, 70%, 75%, 80%,85%, 90%, 95-99%, or above typically demonstrates that a protein is apolymorphic variant, interspecies homolog, or allele of a T1R familymember. Sequence comparison can be performed using any of the sequencecomparison algorithms discussed below. Antibodies that bind specificallyto T1R polypeptides or a conserved region thereof can also be used toidentify alleles, interspecies homologs, and polymorphic variants.

[0052] Polymorphic variants, interspecies homologs, and alleles of T1Rgenes can be confirmed by examining taste-cell-specific expression ofthe putative T1R polypeptide. Typically, T1R polypeptides having anamino acid sequence disclosed herein can be used as a positive controlin comparison to the putative T1R polypeptide to demonstrate theidentification of a polymorphic variant or allele of the T1R familymember. The polymorphic variants, alleles, and interspecies homologs areexpected to retain the seven transmembrane structure of a Gprotein-coupled receptor. For further detail, see WO 00/06592, whichdiscloses related T1R family members, GPCR-B3s, the contents of whichare herein incorporated by reference in a manner consistent with thisdisclosure. GPCR-B3 receptors are referred to herein as rT1R1 and mT1R1.Additionally, see WO 00/06593, which also discloses related T1R familymembers, GPCR-B4s, the contents of which are herein incorporated byreference in a manner consistent with this disclosure. GPCR-B4 receptorsare referred to herein as rT1R2 and mT1R2.

[0053] Nucleotide and amino acid sequence information for T1R familymembers may also be used to construct models of taste-cell-specificpolypeptides in a computer system. These models can be subsequently usedto identify compounds that can activate or inhibit T1R receptorproteins. Such compounds that modulate the activity of T1R familymembers can then be used to investigate the role of T1R genes andreceptors in taste transduction.

[0054] The present invention also provides assays, preferably highthroughput assays, to identify molecules that interact with and/ormodulate a T1R polypeptide. In numerous assays, a particular domain of aT1R family member is used, e.g., an extracellular, transmembrane, orintracellular domain or region. In numerous embodiments, anextracellular domain, transmembrane region or combination thereof may bebound to a solid substrate, and used, e.g., to isolate ligands,agonists, antagonists, or any other molecules that can bind to and/ormodulate the activity of a T1R polypeptide.

[0055] In one aspect of the invention, a new human GPCR gene of the T1Rfamily, termed hT1R3, is provided. The hT1R3 gene was identified fromthe human genome sequence database including the HTGS division ofGenBank. The nucleotide and conceptually translated amino acid sequencefor hT1R3 are provided in SEQ. ID NOS 1-4. The hT1R3 receptor wasidentified in the partially sequenced BAC genomic clone RP5-890O3(database accession number AL139287) by virtue of its sequencesimilarity to the candidate rat taste receptor rT1R1 (accession numberAF127389). By reference, the pairwise identity between the predictedhT1R3 and rT1R1 protein sequences is approximately 34%. Sequencecomparisons with additional members of the GPCR Family C (which includesthe calcium-sensing receptors, putative V2R pheromone receptors, GABA-Breceptors, fish taste receptors, and metabotropic glutamate receptors)indicate that hT1R3 is likely to belong to the Family C subgroup definedby T1R1 and a second rat candidate taste receptor (rT1R2, accessionnumber AF127390).

[0056] The invention also provides the human ortholog, termed hT1R1, ofa rat taste receptor, designated rT1R1. The gene products of rT1R1 andhT1R1 are approximately 74% identical. The mouse gene, mT1R1 has beenreported, see Hoon et al., Cell, 96:541-551 (2000), and maps to achromosomal interval homologous to the interval containing hT1R1. Thenucleotide and conceptually-translated hT1R1 sequences are describedherein as SEQ. ID NOS 15 and 16, respectively.

[0057] While not wishing to be bound to any particular theory, the T1Rfamily of receptors is predicted to be involved in sweet tastetransduction by virtue of the linkage of mTIR3 to the Sac locus, a locuson the distal end of chromosome four that influences sweet taste. HumanT1R3 has also been reported to localize to 1p36.2-1p36.33, a region thatdisplays conserved synteny with the mouse interval containing Sac andT1R1. However, T1R type receptors may mediate other taste modalities,such as bitter, umami, sour and salty.

[0058] Various conservative mutations and substitutions are envisionedto be within the scope of the invention. For instance, it would bewithin the level of skill in the art to perform amino acid substitutionsusing known protocols of recombinant gene technology including PCR, genecloning, site-directed mutagenesis of cDNA, transfection of host cells,and in-vitro transcription. The variants could then be screened fortaste-cell-specific GPCR functional activity.

A. Identification and Characterization of T1R Polypeptides

[0059] The amino acid sequences of the T1R proteins and polypeptides ofthe invention can be identified by putative translation of the codingnucleic acid sequences. These various amino acid sequences and thecoding nucleic acid sequences may be compared to one another or to othersequences according to a number of methods.

[0060] For example, in sequence comparison, typically one sequence actsas a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, as described below for theBLASTN and BLASTP programs, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

[0061] A “comparison window,” as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

[0062] A preferred example of an algorithm that is suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol.215:403-410 (1990), respectively. Software, for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., Altschul etal., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J Mol.Biol. 215:403-410 (1990)). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

[0063] Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a so-called “tree” or “dendogram”showing the clustering relationships used to create the alignment (see,e.g., FIG. 2). PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395 (1984) encoded by the genes were derived by conceptualtranslation of the corresponding open reading frames. Comparison ofthese protein sequences to all known proteins in the public sequencedatabases using BLASTP algorithm revealed their strong homology to themembers of the T1R family, each of the T1R family sequences having atleast about 35 to 50%, and preferably at least 55%, at least 60%, atleast 65%, and most preferably at least 70%, amino acid identity to atleast one known member of the family.

B. Definitions

[0064] As used herein, the following terms have the meanings ascribed tothem unless specified otherwise.

[0065] “Taste cells” include neuroepithelial cells that are organizedinto groups to form taste buds of the tongue, e.g., foliate, fungiform,and circumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329-353 (1989)). Taste cells are also found in the palate and othertissues, such as the esophagus and the stomach.

[0066] “T1R” refers to one or more members of a family of Gprotein-coupled receptors that are expressed in taste cells such asfoliate, fungiform, and circumvallate cells, as well as cells of thepalate, and esophagus (see, e.g., Hoon et al., Cell, 96:541-551 (1999),herein incorporated by reference in its entirety). Members of thisfamily are also referred to as GPCR-B3 and TR1 in WO 00/06592 as well asGPCR-B4 and TR2 in WO 00/06593. GPCR-B3 is also herein referred to asrT1R1, and GPCR-B4 is referred to as rT1R2. Taste receptor cells canalso be identified on the basis of morphology (see, e.g., Roper, supra),or by the expression of proteins specifically expressed in taste cells.T1R family members may have the ability to act as receptors for sweettaste transduction, or to distinguish between various other tastemodalities.

[0067] “T1R” nucleic acids encode a family of GPCRs with seventransmembrane regions that have “G protein-coupled receptor activity,”e.g., they may bind to G proteins in response to extracellular stimuliand promote production of second messengers such as IP3, cAMP, cGMP, andCa²⁺ via stimulation of enzymes such as phospholipase C and adenylatecyclase (for a description of the structure and function of GPCRs, see,e.g., Fong, supra, and Baldwin, supra). A single taste cell may containmany distinct T1R polypeptides.

[0068] The term “T1R” family therefore refers to polymorphic variants,alleles, mutants, and interspecies homologs that: (1) have at leastabout 35 to 50% amino acid sequence identity, optionally about 60, 75,80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQID NOS: 4, 10, 12, 14, or 17, over a window of about 25 amino acids,optionally 50-100 amino acids; (2) specifically bind to antibodiesraised against an immunogen comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 4, 10, 12, 14, 17, andconservatively modified variants thereof; (3) are encoded by a nucleicacid molecule which specifically hybridize (with a size of at leastabout 100, optionally at least about 500-1000 nucleotides) understringent hybridization conditions to a sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 9, 11, 13, 15, 16, 20, andconservatively modified variants thereof, (4) comprise a sequence atleast about 35 to 50% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 4, 10, 12, 14, or 17; or (5) areamplified by primers that specifically hybridize under stringenthybridization conditions to the same sequence as degenerate primer setsencoding SEQ ID NOS: 7, 8, and conservatively modified variants thereof.

[0069] Topologically, certain chemosensory GPCRs have an “N-terminaldomain;” “extracellular domains;” “transmembrane domains” comprisingseven transmembrane regions, and corresponding cytoplasmic, andextracellular loops; “cytoplasmic domains,” and a “C-terminal domain”(see, e.g., Hoon et al., Cell, 96:541-551 (1999); Buck & Axel, Cell,65:175-187 (1991)). These domains can be structurally identified usingmethods known to those of skill in the art, such as sequence analysisprograms that identify hydrophobic and hydrophilic domains (see, e.g.,Stryer, Biochemistry, (3rd ed. 1988); see also any of a number ofInternet based sequence analysis programs, such as those found atdot.imgen.bcm.tmc.edu). Such domains are useful for making chimericproteins and for in vitro assays of the invention, e.g., ligand-bindingassays.

[0070] “Extracellular domains” therefore refers to the domains of T1Rpolypeptides that protrude from the cellular membrane and are exposed tothe extracellular face of the cell. Such domains generally include the“N terminal domain” that is exposed to the extracellular face of thecell, and optionally can include portions of the extracellular loops ofthe transmembrane domain that are exposed to the extracellular face ofthe cell, i.e., the loops between transmembrane regions 2 and 3, betweentransmembrane regions 4 and 5, and between transmembrane regions 6 and7.

[0071] The “N terminal domain” region starts at the N-terminus andextends to a region close to the start of the transmembrane domain. Moreparticularly, in one embodiment of the invention, this domain starts atthe N-terminus and ends approximately at the conserved glutamic acid atamino acid position 563 plus or minus approximately 20 amino acid. Theregion corresponding to amino acids 1-580 of SEQ ID 40 is a particularembodiment of an extracellular domain that extends slightly into thetransmembrane domain. These extracellular domains are useful for invitro ligand-binding assays, both soluble and solid phase. In addition,transmembrane regions, described below, can also bind ligand either incombination with the extracellular domain, and are therefore also usefulfor in vitro ligand-binding assays.

[0072] “Transmembrane domain,” which comprises the seven “transmembraneregions,” refers to the domain of T1R polypeptides that lies within theplasma membrane, and may also include the corresponding cytoplasmic(intracellular) and extracellular loops. In one embodiment, this regioncorresponds to the domain of T1R family members which startsapproximately at the conserved glutamic acid residue at amino acidposition 563 plus or minus 20 amino acids and ends approximately at theconserved tyrosine amino acid residue at position 812 plus or minusapproximately 10 amino acids. The seven transmembrane regions andextracellular and cytoplasmic loops can be identified using standardmethods, as described in Kyte & Doolittle, J. Mol. Biol., 157:105-32(1982)), or in Stryer, supra.

[0073] “Cytoplasmic domains” refers to the domains of T1R polypeptidesthat face the inside of the cell, e.g., the “C terminal domain” and theintracellular loops of the transmembrane domain, e.g., the intracellularloop between transmembrane regions 1 and 2, the intracellular loopbetween transmembrane regions 3 and 4, and the intracellular loopbetween transmembrane regions 5 and 6. “C terminal domain” refers to theregion that spans the end of the last transmembrane domain and theC-terminus of the protein, and which is normally located within thecytoplasm. In one embodiment, this region starts at the conservedtyrosine amino acid residue at position 812 plus or minus approximately10 amino acids and continues to the C-terminus of the polypeptide.

[0074] The term “ligand-binding region” or “ligand-binding domain”refers to sequences derived from a chemosensory receptor, particularly ataste receptor, that substantially incorporates at least theextracellular domain of the receptor. In one embodiment, theextracellular domain of the ligand-binding region may include theN-terminal domain and, optionally, portions of the transmembrane domain,such as the extracellular loops of the transmembrane domain. Theligand-binding region may be capable of binding a ligand, and moreparticularly, a tastant.

[0075] The phrase “functional effects” in the context of assays fortesting compounds that modulate T1R family member mediated tastetransduction includes the determination of any parameter that isindirectly or directly under the influence of the receptor, e.g.,functional, physical and chemical effects. It includes ligand binding,changes in ion flux, membrane potential, current flow, transcription, Gprotein binding, GPCR phosphorylation or dephosphorylation, signaltransduction, receptor-ligand interactions, second messengerconcentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro,in vivo, and ex vivo and also includes other physiologic effects suchincreases or decreases of neurotransmitter or hormone release.

[0076] By “determining the functional effect” in the context of assaysis meant assays for a compound that increases or decreases a parameterthat is indirectly or directly under the influence of a T1R familymember, e.g., functional, physical and chemical effects. Such functionaleffects can be measured by any means known to those skilled in the art,e.g., changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, oocyte T1R gene expression; tissue culture cell T1Rexpression; transcriptional activation of T1R genes; ligand-bindingassays; voltage, membrane potential and conductance changes; ion fluxassays; changes in intracellular second messengers such as cAMP, cGMP,and inositol triphosphate (IP3); changes in intracellular calciumlevels; neurotransmitter release, and the like.

[0077] “Inhibitors,” “activators,” and “modulators” of T1R genes orproteins are used interchangeably to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays fortaste transduction, e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activators are compounds that, e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize, or up regulate taste transduction, e.g.,agonists. Modulators include compounds that, e.g., alter the interactionof a receptor with: extracellular proteins that bind activators orinhibitor (e.g., ebnerin and other members of the hydrophobic carrierfamily); G proteins; kinases (e.g., homologs of rhodopsin kinase andbeta adrenergic receptor kinases that are involved in deactivation anddesensitization of a receptor); and arresting, which also deactivate anddesensitize receptors. Modulators can include genetically modifiedversions of T1R family members, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for inhibitors andactivators include, e.g., expressing T1R family members in cells or cellmembranes, applying putative modulator compounds, in the presence orabsence of tastants, e.g., sweet tastants, and then determining thefunctional effects on taste transduction, as described above. Samples orassays comprising T1R family members that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofmodulation. Control samples (untreated with modulators) are assigned arelative T1R activity value of 100%. Inhibition of a T1R is achievedwhen the T1R activity value relative to the control is about 80%,optionally 50% or 25-0%. Activation of a T1R is achieved when the T1Ractivity value relative to the control is 110%, optionally 150%,optionally 200-500%, or 1000-3000% higher.

[0078] The terms “purified,” “substantially purified,” and “isolated” asused herein refer to the state of being free of other, dissimilarcompounds with which the compound of the invention is normallyassociated in its natural state, so that the “purified,” “substantiallypurified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%,or 20%, and most preferably at least 50% or 75% of the mass, by weight,of a given sample. In one preferred embodiment, these terms refer to thecompound of the invention comprising at least 95% of the mass, byweight, of a given sample. As used herein, the terms “purified,”“substantially purified,” and “isolated” “isolated,” when referring to anucleic acid or protein, of nucleic acids or proteins, also refers to astate of purification or concentration different than that which occursnaturally in the mammalian, especially human, body. Any degree ofpurification or concentration greater than that which occurs naturallyin the mammalian, especially human, body, including (1) the purificationfrom other associated structures or compounds or (2) the associationwith structures or compounds to which it is not normally associated inthe mammalian, especially human, body, are within the meaning of“isolated.” The nucleic acid or protein or classes of nucleic acids orproteins, described herein, may be isolated, or otherwise associatedwith structures or compounds to which they are not normally associatedin nature, according to a variety of methods and processes known tothose of skill in the art.

[0079] As used herein, the term “isolated,” when referring to a nucleicacid or polypeptide refers to a state of purification or concentrationdifferent than that which occurs naturally in the mammalian, especiallyhuman, body. Any degree of purification or concentration greater thanthat which occurs naturally in the body, including (1) the purificationfrom other naturally-occurring associated structures or compounds, or(2) the association with structures or compounds to which it is notnormally associated in the body are within the meaning of “isolated” asused herein. The nucleic acids or polypeptides described herein may beisolated or otherwise associated with structures or compounds to whichthey are not normally associated in nature, according to a variety ofmethods and processed known to those of skill in the art.

[0080] As used herein, the terms “amplifying” and “amplification” referto the use of any suitable amplification methodology for generating ordetecting recombinant or naturally expressed nucleic acid, as describedin detail, below. For example, the invention provides methods andreagents (e.g., specific degenerate oligonucleotide primer pairs) foramplifying (e.g., by polymerase chain reaction, PCR) naturally expressed(e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of theinvention (e.g., tastant-binding sequences of the invention) in vivo orin vitro.

[0081] The term “7-transmembrane receptor” means a polypeptide belongingto a superfamily of transmembrane proteins that have seven domains thatspan the plasma membrane seven times (thus, the seven domains are called“transmembrane” or “TM” domains TM I to TM VII). The families ofolfactory and certain taste receptors each belong to this super-family.7-transmembrane receptor polypeptides have similar and characteristicprimary, secondary and tertiary structures, as discussed in furtherdetail below.

[0082] The term “library” means a preparation that is a mixture ofdifferent nucleic acid or polypeptide molecules, such as the library ofrecombinantly generated chemosensory, particularly taste receptorligand-binding domains generated by amplification of nucleic acid withdegenerate primer pairs, or an isolated collection of vectors thatincorporate the amplified ligand-binding domains, or a mixture of cellseach randomly transfected with at least one vector encoding an tastereceptor.

[0083] The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones (see e.g., Oligonucleotides and Analogues, a PracticalApproach, ed. F. Eckstein, Oxford Univ. Press (1991); AntisenseStrategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds.Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993);Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997);Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, AntisenseNucleic Acid Drug Dev, 6:153-156 (1996)).

[0084] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating, e.g., sequences inwhich the third position of one or more selected codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0085] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0086] The term “plasma membrane translocation domain” or simply“translocation domain” means a polypeptide domain that, whenincorporated into the amino terminus of a polypeptide coding sequence,can with great efficiency “chaperone” or “translocate” the hybrid(“fusion”) protein to the cell plasma membrane. For instance, a“translocation domain” may be derived from the amino terminus of thebovine rhodopsin receptor polypeptide, a 7-transmembrane receptor.However, rhodopsin from any mammal may be used, as can othertranslocation facilitating sequences. Thus, the translocation domain isparticularly efficient in translocating 7-transmembrane fusion proteinsto the plasma membrane, and a protein (e.g., a taste receptorpolypeptide) comprising an amino terminal translocating domain will betransported to the plasma membrane more efficiently than without thedomain. However, if the N-terminal domain of the polypeptide is activein binding, the use of other translocation domains may be preferred.

[0087] The “translocation domain,” “ligand-binding domain”, and chimericreceptors compositions described herein also include “analogs,” or“conservative variants” and “mimetics” (“peptidomimetics”) withstructures and activity that substantially correspond to the exemplarysequences. Thus, the terms “conservative variant” or “analog” or“mimetic” refer to a polypeptide which has a modified amino acidsequence, such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity, asdefined herein. These include conservatively modified variations of anamino acid sequence, i.e., amino acid substitutions, additions ordeletions of those residues that are not critical for protein activity,or substitution of amino acids with residues having similar properties(e.g., acidic, basic, positively or negatively charged, polar ornon-polar, etc.) such that the substitutions of even critical aminoacids does not substantially alter structure and/or activity.

[0088] More particularly, “conservatively modified variants” applies toboth amino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein.

[0089] For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.

[0090] Such nucleic acid variations are “silent variations,” which areone species of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0091] Conservative substitution tables providing functionally similaramino acids are well known in the art. For example, one exemplaryguideline to select conservative substitutions includes (originalresidue followed by exemplary substitution): ala/gly or ser; arg/lys;asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro;his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu;met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr;tyr/trp or phe; val/ile or leu. An alternative exemplary guideline usesthe following six groups, each containing amino acids that areconservative substitutions for one another: 1) Alanine (A), Serine (S),Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine(N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H.Freeman and Company (1984); Schultz and Schimer, Principles of ProteinStructure, Springer-Vrlag (1979)). One of skill in the art willappreciate that the above-identified substitutions are not the onlypossible conservative substitutions. For example, for some purposes, onemay regard all charged amino acids as conservative substitutions foreach other whether they are positive or negative. In addition,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence can also be considered “conservatively modifiedvariations.”

[0092] The terms “mimetic” and “peptidomimetic” refer to a syntheticchemical compound that has substantially the same structural and/orfunctional characteristics of the polypeptides, e.g., translocationdomains, ligand-binding domains, or chimeric receptors of the invention.The mimetic can be either entirely composed of synthetic, non-naturalanalogs of amino acids, or may be a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also incorporate any amount of natural amino acidconservative substitutions as long as such substitutions also do notsubstantially alter the mimetic's structure and/or activity.

[0093] As with polypeptides of the invention which are conservativevariants, routine experimentation will determine whether a mimetic iswithin the scope of the invention, i.e., that its structure and/orfunction is not substantially altered. Polypeptide mimetic compositionscan contain any combination of non-natural structural components, whichare typically from three structural groups: a) residue linkage groupsother than the natural amide bond (“peptide bond”) linkages; b)non-natural residues in place of naturally occurring amino acidresidues; or c) residues which induce secondary structural mimicry,i.e., to induce or stabilize a secondary structure, e.g., a beta turn,gamma turn, beta sheet, alpha helix conformation, and the like. Apolypeptide can be characterized as a mimetic when all or some of itsresidues are joined by chemical means other than natural peptide bonds.Individual peptidomimetic residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola, Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide Backbone Modifications,” Marcell Dekker, NY (1983)). Apolypeptide can also be characterized as a mimetic by containing all orsome non-natural residues in place of naturally occurring amino acidresidues; non-natural residues are well described in the scientific andpatent literature.

[0094] A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

[0095] A “labeled nucleic acid probe or oligonucleotide” is one that isbound, either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

[0096] As used herein a “nucleic acid probe or oligonucleotide” isdefined as a nucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

[0097] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0098] A “promoter” is defined as an array of nucleic acid sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

[0099] As used herein, “recombinant” refers to a polynucleotidesynthesized or otherwise manipulated in vitro (e.g., “recombinantpolynucleotide”), to methods of using recombinant polynucleotides toproduce gene products in cells or other biological systems, or to apolypeptide (“recombinant protein”) encoded by a recombinantpolynucleotide. “Recombinant means” also encompass the ligation ofnucleic acids having various coding regions or domains or promotersequences from different sources into an expression cassette or vectorfor expression of, e.g., inducible or constitutive expression of afusion protein comprising a translocation domain of the invention and anucleic acid sequence amplified using a primer of the invention.

[0100] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0101] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridisation with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,S×SSC, and 1% SDS, incubating at 42° C., or, S×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Suchhybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10,15, 30, 60; or more minutes.

[0102] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially related if the polypeptides whichthey encode are substantially related. This occurs, for example, when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

[0103] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0104] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

[0105] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0106] An “anti-T1R” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a T1R gene, cDNA, or asubsequence thereof.

[0107] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

[0108] The phrase “specifically (or selectively) binds” to an antibodyor, “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to a T1R family member fromspecific species such as rat, mouse, or human can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith the T1R polypeptide or an immunogenic portion thereof and not withother proteins, except for orthologs or polymorphic variants and allelesof the T1R polypeptide. This selection may be achieved by subtractingout antibodies that cross-react with T1R molecules from other species orother T1R molecules. Antibodies can also be selected that recognize onlyT1R GPCR family members but not GPCRs from other families. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual, (1988), for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

[0109] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0110] The term “expression vector” refers to any recombinant expressionsystem for the purpose of expressing a nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including prokaryotic, yeast, fungal, plant, insect or mammalian cell.The term includes linear or circular expression systems. The termincludes expression systems that remain episomal or integrate into thehost cell genome. The expression systems can have the ability toself-replicate or not, i.e., drive only transient expression in a cell.The term includes recombinant expression “cassettes which contain onlythe minimum elements needed for transcription of the recombinant nucleicacid.

[0111] By “host cell” is meant a cell that contains an expression vectorand supports the replication or expression of the expression vector.Host cells may be prokaryotic cells such as E. toll, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

C. Isolation and Expression of T1R Polypeptides

[0112] Isolation and expression of the T1Rs, or fragments or variantsthereof, of the invention can be performed as described below. PCRprimers can be used for the amplification of nucleic acids encodingtaste receptor ligand-binding regions, and libraries of these nucleicacids can optionally be generated. Individual expression vectors orlibraries of expression vectors can then be used to infect or transfecthost cells for the functional expression of these nucleic acids orlibraries. These genes and vectors can be made and expressed in vitro orin vivo. One of skill will recognize that desired phenotypes foraltering and controlling nucleic acid expression can be obtained bymodulating the expression or activity of the genes and nucleic acids(e.g., promoters, enhancers and the like) within the vectors of theinvention. Any of the known methods described for increasing ordecreasing expression or activity can be used. The invention can bepracticed in conjunction with any method or protocol known in the art,which are well described in the scientific and patent literature.

[0113] The nucleic acid sequences of the invention and other nucleicacids used to practice this invention, whether RNA, cDNA, genomic DNA,vectors, viruses or hybrids thereof, may be isolated from a variety ofsources, genetically engineered, amplified, and/or expressedrecombinantly. Any recombinant expression system can be used, including,in addition to mammalian cells, e.g., bacterial, yeast, insect, or plantsystems.

[0114] Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982);Adams, Am. Chem. Soc. 105:661 (1983); Belousov, Nucleic Acids Res.25:3440-3444 (1997); Frenkel, Free Radic. Biol. Med. 19:373-380 (1995);Blommers, Biochemistry 33:7886-7896 (1994); Narang, Meth. Enzymol. 68:90(1979); Brown, Meth. Enzymol. 68:109 (1979); Beaucage, Tetra. Lett.22:1859 (1981); U.S. Pat. No. 4,458,066. Double-stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

[0115] Techniques for the manipulation of nucleic acids, such as, forexample, for generating mutations in sequences, subcloning, labelingprobes, sequencing, hybridization and the like are well described in thescientific and patent literature. See, e.g., Sambrook, ed., MolecularCloning: a Laboratory manual (2nd ed.), Vols. 1-3, Cold Spring HarborLaboratory (1989); Current Protocols in Molecular Biology, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I, Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993).

[0116] Nucleic acids, vectors, capsids, polypeptides, and the like canbe analyzed and quantified by any of a number of general means wellknown to those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatography, various immunological methods, e.g.,fluid or gel precipitin reactions, immunodiffusion,immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immuno-fluorescent assays, Southernanalysis, Northern analysis, dot-blot analysis, gel electrophoresis(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or targetor signal amplification methods, radiolabeling, scintillation counting,and affinity chromatography.

[0117] Oligonucleotide primers may be used to amplify nucleic acidfragments encoding taste receptor ligand-binding regions. The nucleicacids described herein can also be cloned or measured quantitativelyusing amplification techniques. Amplification methods are also wellknown in the art, and include, e.g., polymerase chain reaction, PCR (PCRProtocols, a Guide to Methods and Applications, ed. Innis. AcademicPress, N.Y. (1990) and PCR Strategies, ed. Innis, Academic Press, Inc.,N.Y. (1995), ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560(1989); Landegren, Science 241:1077,(1988); Barringer, Gene 89:117(1990)); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad.Sci. USA 86:1173 (1989)); and, self-sustained sequence replication (see,e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87:1874 (1990)); Q Betareplicase amplification (see, e.g., Smith, J. Clin. Microbiol.35:1477-1491 (1997)); automated Q-beta replicase amplification assay(see, e.g., Burg, Mol. Cell. Probes 10:257-271 (1996)) and other RNApolymerase mediated techniques (e.g., NASBA, Cangene, Mississauga,Ontario); see also Berger, Methods Enzymol. 152:307-316 (1987);Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan,Biotechnology 13:563-564 (1995). The primers can be designed to retainthe original sequence of the “donor” 7-membrane receptor. Alternatively,the primers can encode amino acid residues that are conservativesubstitutions (e.g., hydrophobic for hydrophobic residue, see abovediscussion) or functionally benign substitutions (e.g., do not preventplasma membrane insertion, cause cleavage by peptidase, cause abnormalfolding of receptor, and the like). Once amplified, the nucleic acids,either individually or as libraries, may be cloned according to methodsknown in the art, if desired, into any of a variety of vectors usingroutine molecular biological methods; methods for cloning in vitroamplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039.

[0118] The primer pairs may be designed to selectively amplifyligand-binding regions of the T1R family members. These regions may varyfor different ligands or tastants. Thus, what may be a minimal bindingregion for one tastant, may be too limiting for a second tastant.Accordingly, ligand-binding regions of different sizes comprisingdifferent extracellular domain structures may be amplified.

[0119] Paradigms to design degenerate primer pairs are well known in theart. For example, a COnsensus-DEgenerate Hybrid Oligonucleotide Primer(CODEHOP) strategy computer program is accessible ashttp://blocks.fhcrc.org/codehop.html, and is directly linked from theBlockMaker multiple sequence alignment site for hybrid primer predictionbeginning with a set of related protein sequences, as known tastereceptor ligand-binding regions (see, e.g., Rose, Nucleic Acids Res.26:1628-1635 (1998); Singh, Biotechniques 24:318-319 (1998)).

[0120] Means to synthesize oligonucleotide primer pairs are well knownin the art. “Natural” base pairs or synthetic base pairs can be used.For example, use of artificial nucleobases offers a versatile approachto manipulate primer sequence and generate a more complex mixture ofamplification products. Various families of artificial nucleobases arecapable of assuming multiple hydrogen bonding orientations throughinternal bond rotations to provide a means for degenerate molecularrecognition. Incorporation of these analogs into a single position of aPCR primer allows for generation of a complex library of amplificationproducts. See, e.g., Hoops, Nucleic Acids Res. 25:4866-4871 (1997).Nonpolar molecules can also be used to mimic the shape of natural DNAbases. A non-hydrogen-bonding shape mimic for adenine can replicateefficiently and selectively against a nonpolar shape mimic for thymine(see, e.g., Morales, Nat. Struct. Biol. 5:950-954 (1998)). For example,two degenerate bases can be the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one or the purine baseN6-methoxy-2,6-diaminopurine (see, e.g., Hill, Proc. Natl. Acad. Sci.USA 95:4258-4263 (1998)). Exemplary degenerate primers of the inventionincorporate the nucleobase analog5′-Dimethoxytrityl-N-benzoyl-2′-deoxy-Cytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (the term “P” inthe sequences, see above). This pyrimidine analog hydrogen bonds withpurines, including A and G residues.

[0121] Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a taste receptor disclosed herein can beisolated using the nucleic acid probes described above. Alternatively,expression libraries can be used to clone T1R polypeptides andpolymorphic variants, alleles, and interspecies homologs thereof, bydetecting expressed homologs immunologically with antisera or purifiedantibodies made against a T1R polypeptide, which also recognize andselectively bind to the T1R homolog.

[0122] Nucleic acids that encode ligand-binding regions of tastereceptors may be generated by amplification (e.g., PCR) of appropriatenucleic acid sequences using degenerate primer pairs. The amplifiednucleic acid can be genomic DNA from any cell or tissue or mRNA or cDNAderived from taste receptor-expressing cells.

[0123] In one embodiment, hybrid protein-coding sequences comprisingnucleic acids encoding T1Rs fused to a translocation sequences may beconstructed. Also provided are hybrid T1Rs comprising the translocationmotifs and tastant-binding domains of other families of chemosensoryreceptors, particularly taste receptors. These nucleic acid sequencescan be operably linked to transcriptional or translational controlelements, e.g., transcription and translation initiation sequences,promoters and enhancers, transcription and translation terminators,polyadenylation sequences, and other sequences useful for transcribingDNA into RNA. In construction of recombinant expression cassettes,vectors, and transgenics, a promoter fragment can be employed to directexpression of the desired nucleic acid in all desired cells or tissues.

[0124] In another embodiment, fusion proteins may include C-terminal orN-terminal translocation sequences. Further, fusion proteins cancomprise additional elements, e.g., for protein detection, purification,or other applications. Detection and purification facilitating domainsinclude, e.g., metal chelating peptides such as polyhistidine tracts,histidine-tryptophan modules, or other domains that allow purificationon immobilized metals; maltose binding protein; protein A domains thatallow purification on immobilized immunoglobulin; or the domain utilizedin the FLAGS extension/affinity purification system (Immunex Corp,Seattle Wash.).

[0125] The inclusion of a cleavable linker sequences such as Factor Xa(see, e.g., Ottavi, Biochimie 80:289-293 (1998)), subtilisin proteaserecognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997));enterokinase (Invitrogen, San Diego, Calif.), and the like, between thetranslocation domain (for efficient plasma membrane expression) and therest of the newly translated polypeptide may be useful to facilitatepurification. For example, one construct can include a polypeptideencoding a nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin, an enterokinase cleavage site (see, e.g.,Williams, Biochemistry 34:1787-1797 (1995)), and an C-terminaltranslocation domain. The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the desired protein(s) from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see, e.g., Kroll, DNA Cell. Biol. 12:441-53 (1993).

[0126] Expression vectors, either as individual expression vectors or aslibraries of expression vectors, comprising the ligand-binding domainencoding sequences may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. See,e.g., Roberts, Nature 328:731 (1987); Berger supra; Schneider, ProteinExpr. Purif. 6435:10 (1995); Sambrook; Tijssen; Ausubel. Productinformation from manufacturers of biological reagents and experimentalequipment also provide information regarding known biological methods.The vectors can be isolated from natural sources, obtained from suchsources as ATCC or GenBank libraries, or prepared by synthetic orrecombinant methods.

[0127] The nucleic acids can be expressed in expression cassettes,vectors or viruses which are stably or transiently expressed in cells(e.g., episomal expression systems). Selection markers can beincorporated into expression cassettes and vectors to confer aselectable phenotype on transformed cells and sequences. For example,selection markers can code for episomal maintenance and replication suchthat integration into the host genome is not required. For example, themarker may encode antibiotic resistance (e.g., chloramphenicol,kanamycin, G418, bleomycin, hygromycin) or herbicide resistance (e.g.,chlorosulfuron or Basta) to permit selection of those cells transformedwith the desired DNA sequences (see, e.g., Blondelet-Rouault, Gene190:315-317 (1997); Aubrecht, J. Pharmacol. Exp. Ther. 281:992-997(1997)). Because selectable marker genes conferring resistance tosubstrates like neomycin or hygromycin can only be utilized in tissueculture, chemoresistance genes are also used as selectable markers invitro and in vivo.

[0128] A chimeric nucleic acid sequence may encode a T1R ligand-bindingdomain within any 7-transmembrane polypeptide. Because 7-transmembranereceptor polypeptides have similar primary sequences and secondary andtertiary structures, structural domains (e.g., extracellular domain, TMdomains, cytoplasmic domain, etc.) can be readily identified by sequenceanalysis. For example, homology modeling, Fourier analysis and helicalperiodicity detection can identify and characterize the seven domainswith a 7-transmembrane receptor sequence. Fast Fourier Transform (FFT)algorithms can be used to assess the dominant periods that characterizeprofiles of the hydrophobicity and variability of analyzed sequences.Periodicity detection enhancement and alpha helical periodicity indexcan be done as by, e.g., Donnelly, Protein Sci. 2:55-70 (1993). Otheralignment and modeling algorithms are well known in the art, see, e.g.,Peitsch, Receptors Channels 4:161-164 (1996); Kyte & Doolittle, J. Md.Bio., 157:105-132 (1982); Cronet, Protein Eng. 6:59-64 (1993) (homologyand “discover modeling”); http://bioinfo.weizmann.ac.il/.

[0129] The present invention also includes not only the DNA and proteinshaving the specified nucleic and amino acid sequences, but also DNAfragments, particularly fragments of, e.g., 40, 60, 80, 100, 150, 200,or 250 nucleotides, or more, as well as protein fragments of, e.g., 10,20, 30, 50, 70, 100, or 150 amino acids, or more. Optionally, thenucleic acid fragments can encode an antigenic polypeptide which iscapable of binding to an antibody raised against a T1R family member.Further, a protein fragment of the invention can optionally be anantigenic fragment which is capable of binding to an antibody raisedagainst a T1R family member.

[0130] Also contemplated are chimeric proteins, comprising at least 10,20, 30, 50, 70, 100, or 150 amino acids, or more, of one of at least oneof the T1R polypeptides described herein, coupled to additional aminoacids representing all or part of another GPCR, preferably a member ofthe 7 transmembrane superfamily. These chimeras can be made from theinstant receptors and another GPCR, or they can be made by combining twoor more of the present receptors. In one embodiment, one portion of thechimera corresponds tom or is derived from the extracellular domain of aT1R polypeptide of the invention. In another embodiment, one portion ofthe chimera corresponds to, or is derived from the extracellular domainand one or more of the transmembrane domains of a T1R polypeptidedescribed herein, and the remaining portion or portions can come fromanother GPCR. Chimeric receptors are well known in the art, and thetechniques for creating them and the selection and boundaries of domainsor fragments of G protein-coupled receptors for incorporation thereinare also well known. Thus, this knowledge of those skilled in the artcan readily be used to create such chimeric receptors. The use of suchchimeric receptors can provide, for example, a taste selectivitycharacteristic of one of the receptors specifically disclosed herein,coupled with the signal transduction characteristics of anotherreceptor, such as a well known receptor used in prior art assay systems.

[0131] For example, a domain such as a ligand-binding domain, anextracellular domain, a transmembrane domain, a transmembrane domain, acytoplasmic domain, an N-terminal domain, a C-terminal domain, or anycombination thereof, can be covalently linked to a heterologous protein.For instance, an T1R extracellular domain can be linked to aheterologous GPCR transmembrane domain, or a heterologous GPCRextracellular domain can be linked to a T1R transmembrane domain. Otherheterologous proteins of choice can include, e.g., green fluorescentprotein, β-gal, glutamtate receptor, and the rhodopsin presequence.

[0132] Also within the scope of the invention are host cells forexpressing the T1Rs, fragments, or variants of the invention. To obtainhigh levels of expression of a cloned gene or nucleic acid, such ascDNAs encoding the T1Rs, fragments, or variants of the invention, one ofskill typically subclones the nucleic acid sequence of interest into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. However, bacterial oreukaryotic expression systems can be used.

[0133] Any of the well known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook et al.) It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at lest one nucleic acid molecule into the host cell capableof expressing the T1R, fragment, or variant of interest.

[0134] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe receptor, fragment, or variant of interest, which is then recoveredfrom the culture using standard techniques. Examples of such techniquesare well known in the art. See, e.g., WO 00/06593, which is incorporatedby reference in a manner consistent with this disclosure.

D. Detection of T1R Polypeptides

[0135] In addition to the detection of T1R genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect T1Rs, e.g., to identify taste receptor cells, andvariants of T1R family members. Immunoassays can be used toqualitatively or quantitatively analyze the T1Rs. A general overview ofthe applicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

[0136] 1. Antibodies to T1R Family Members

[0137] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with a T1R family member are known to those of skillin the art (see, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles andPractice (2d ed. 1986); and Kohler & Milstein, Nature, 256:495-497(1975)). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science,246:1275-1281 (1989); Ward et al., Nature, 341:544-546 (1989)).

[0138] A number of T1R-comprising immunogens may be used to produceantibodies specifically reactive with a T1R family member. For example,a recombinant T1R polypeptide, or an antigenic fragment thereof, can beisolated as described herein. Suitable antigenic regions include, e.g.,the consensus sequences that are used to identify members of the T1Rfamily. Recombinant proteins can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

[0139] Methods of production of polyclonal antibodies are known to thoseof skill in the art. For example, an inbred strain of mice (e.g., BALB/Cmice) or rabbits is immunized with the protein using a standardadjuvant, such as Freund's adjuvant, and a standard immunizationprotocol. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the T1R. When appropriately high titers of antibody to the immunogenare obtained, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired (see Harlow & Lane,supra).

[0140] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen may be immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol.,6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science, 246:1275-1281 (1989).

[0141] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 104 or greaterare selected and tested for their cross reactivity against non-T1Rpolypeptides, or even other T1R family members or other related proteinsfrom other organisms, using a competitive binding immunoassay. Specificpolyclonal antisera and monoclonal antibodies will usually bind with aKd of at least about 0.1 mM, more usually at least about 1 pM,optionally at least about 0.1 p.M or better, and optionally 0.01 pM orbetter.

[0142] Once T1R family member specific antibodies are available,individual T1R proteins and protein fragments can be detected by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures, see Basic and Clinical Immunology (Stites & Terreds., 7th ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

[0143] 2. Immunological Binding Assays

[0144] T1R proteins, fragments, and variants can be detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). For a review of the general immunoassays, seealso Methods in Cell Biology: Antibodies in Cell Biology, volume 37(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds.,7th ed. 1991). Immunological binding assays (or immunoassays) typicallyuse an antibody that specifically binds to a protein or antigen ofchoice (in this case a T1R family member or an antigenic subsequencethereof). The antibody (e.g., anti-T1R) may be produced by any of anumber of means well known to those of skill in the art and as describedabove.

[0145] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeled T1Rpolypeptide or a labeled anti-T1R antibody. Alternatively, the labelingagent may be a third moiety, such a secondary antibody, thatspecifically binds to the antibody/T1R complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol.,111:1401-1406 (1973); Akerstrom et al., J. Immunol., 135:2589-2542(1985)). The labeling agent can be modified with a detectable moiety,such as biotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

[0146] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, optionally from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0147] a. Non-competitive Assay Formats

[0148] Immunoassays for detecting a T1R polypeptide in a sample may beeither competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of antigen is directly measured. In onepreferred “sandwich” assay, for example, the anti-T1R antibodies can bebound directly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture the T1R polypeptide present in thetest sample. The T1R polypeptide is thus immobilized is then bound by alabeling agent, such as a second T1R antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second or thirdantibody is typically modified with a detectable moiety, such as biotin,to which another molecule specifically binds, e.g., streptavidin, toprovide a detectable moiety.

[0149] b. Competitive Assay Formats

[0150] In competitive assays, the amount of T1R polypeptide present inthe sample is measured indirectly by measuring the amount of a known,added (exogenous) T1R polypeptide displaced (competed away) from ananti-T1R antibody by the unknown T1R polypeptide present in a sample. Inone competitive assay, a known amount of T1R polypeptide is added to asample and the sample is then contacted with an antibody thatspecifically binds to the T1R. The amount of exogenous T1R polypeptidebound to the antibody is inversely proportional to the concentration ofT1R polypeptide present in the sample. In a particularly preferredembodiment, the antibody is immobilized on a solid substrate. The amountof T1R polypeptide bound to the antibody may be determined either bymeasuring the amount of T1R polypeptide present in a T1R/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed protein. The amount of T1R polypeptide may be detected byproviding a labeled T1R molecule.

[0151] A hapten inhibition assay is another preferred competitive assay.In this assay the known T1R polypeptide is immobilized on a solidsubstrate. A known amount of anti-T1R antibody is added to the sample,and the sample is then contacted with the immobilized T1R. The amount ofanti-T1R antibody bound to the known immobilized T1R polypeptide isinversely proportional to the amount of T1R polypeptide present in thesample. Again, the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0152] c. Cross-reactivity Determinations

[0153] Immunoassays in the competitive binding format can also be usedfor cross-reactivity determinations. For example, a protein at leastpartially encoded by the nucleic acid sequences disclosed herein can beimmobilized to a solid support. Proteins (e.g., T1R polypeptides andhomologs) are added to the assay that compete for binding of theantisera to the immobilized antigen. The ability of the added proteinsto compete for binding of the antisera to the immobilized protein iscompared to the ability of the T1R polypeptide encoded by the nucleicacid sequences disclosed herein to compete with itself. The percentcross-reactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% cross-reactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs. In addition, peptides comprising amino acidsequences representing conserved motifs that are used to identifymembers of the T1R family can be used in cross-reactivitydeterminations.

[0154] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps an allele or polymorphic variant of a T1Rfamily member, to the immunogen protein (i.e., T1R polypeptide encodedby the nucleic acid sequences disclosed herein). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by nucleic acidsequences disclosed herein required to inhibit 50% of binding, then thesecond protein is said to specifically bind to the polyclonal antibodiesgenerated to a T1R immunogen.

[0155] Antibodies raised against T1R conserved motifs can also be usedto prepare antibodies that specifically bind only to GPCRs of the T1Rfamily, but not to GPCRs from other families.

[0156] Polyclonal antibodies that specifically bind to a particularmember of the T1R family can be made by subtracting out cross-reactiveantibodies using other T1R family members. Species-specific polyclonalantibodies can be made in a similar way. For example, antibodiesspecific to human T1R1 can be made by, subtracting out antibodies thatare cross-reactive with orthologous sequences, e.g., rat T1R1 or mouseT1R1.

[0157] d. Other Assay Formats

[0158] Western blot (immunoblot) analysis is used to detect and quantifythe presence of T1R polypeptide in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the T1R polypeptide. The anti-T1R polypeptideantibodies specifically bind to the T1R polypeptide on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-T1Rantibodies.

[0159] Other, assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev., 5:34-41 (1986)).

[0160] e. Reduction of Non-specific Binding

[0161] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0162] f. Labels

[0163] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADSTM),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., 3H, 1251, 3sS, 14C, or-³²p), enzymes (e.g., horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0164] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0165] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize a T1R polypeptide, or secondary antibodies that recognizeanti-T1R.

[0166] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0167] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge-coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0168] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

E. Detection of Modulators

[0169] Compositions and methods for determining whether a test compoundspecifically binds to a chemosensory receptor of the invention, both invitro and in vivo, are described below. Many aspects of cell physiologycan be monitored to assess the effect of ligand binding to a T1Rpolypeptide of the invention. These assays may be performed on intactcells expressing a chemosensory receptor, on permeabilized cells, or onmembrane fractions produced by standard methods.

[0170] Taste receptors bind tastants and initiate the transduction ofchemical stimuli into electrical signals. An activated or inhibited Gprotein will in turn alter the properties of target enzymes, channels,and other effector proteins. Some examples are the activation of cGMPphosphodiesterase by transducin in the visual system, adenylate cyclaseby the stimulatory G protein, phospholipase C by Gq and other cognate Gproteins, and modulation of diverse channels by Gi and other G proteins.Downstream consequences can also be examined such as generation ofdiacyl glycerol and IP3 by phospholipase C, and in turn, for calciummobilization by IP3.

[0171] The T1R proteins or polypeptides of the assay will typically beselected from a polypeptide having a sequence of SEQ ID NOS: 4, 10, 12,14, 17, or fragments or conservatively modified variants thereof.Optionally, the fragments and variants can be antigenic fragments andvariants which bind to an anti-T1R antibody.

[0172] Alternatively, the T1R proteins or polypeptides of the assay canbe derived from a eukaryote host cell and can include an amino acidsubsequence having amino acid sequence identity to SEQ ID NOS: 4, 10,12, 14, 17, or fragments or conservatively modified variants thereof.Generally, the amino acid sequence identity will be at least 35 to 50%,or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Optionally, theT1R proteins or polypeptides of the assays can comprise a domain of aT1R protein, such as an extracellular domain, transmembrane region,transmembrane domain, cytoplasmic domain, ligand-binding domain, and thelike. Further, as described above, the T1R protein or a domain thereofcan be covalently linked to a heterologous protein to create a chimericprotein used in the assays described herein.

[0173] Modulators of T1R receptor activity are tested using T1R proteinsor polypeptides as described above, either recombinant or naturallyoccurring. The T1R proteins or polypeptides can be isolated, expressedin a cell, expressed in a membrane derived from a cell, expressed intissue or in an animal, either recombinant or naturally occurring. Forexample, tongue slices, dissociated cells from a tongue, transformedcells, or membranes can be used. Modulation can be tested using one ofthe in vitro or in vivo assays described herein.

[0174] 1. In vitro Binding Assays

[0175] Taste transduction can also be examined in vitro with soluble orsolid state reactions, using a T1R polypeptide of the invention. In aparticular embodiment, a T1R ligand-binding domain can be used in vitroin soluble or solid state reactions to assay for ligand binding.

[0176] For instance, the T1R N-terminal domain is predicted to beinvolved in ligand binding. More particularly, the T1Rs belong to a GPCRsub-family that is characterized by large, approximately 600 amino acid,extracellular N-terminal segments. These N-terminal segments are thoughtto form, at least in part, the ligand-binding domains, and are thereforeuseful in biochemical assays to identify T1R agonists and antagonists.The ligand-binding domain may also contain additional portions of theextracellular domain, such as the extracellular loops of thetransmembrane domain. Similar assays have been used with other GPCRsthat are related to the T1Rs, such as the metabotropic glutamatereceptors (see, e.g., Han and Hampson, J. Biol. Chem. 274:10008-10013(1999)). These assays might involve displacing a radioactively orfluorescently labeled ligand, measuring changes in intrinsicfluorescence or changes in proteolytic susceptibility, etc.

[0177] Ligand binding to a T1R polypeptide of the invention can betested in solution, in a bilayer membrane, optionally attached to asolid phase, in a lipid monolayer, or in vesicles. Binding of amodulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index)hydrodynamic (e.g., shape), chromatographic, or solubility properties.Preferred binding assays of the invention are biochemical binding assaysthat use recombinant soluble N-terminal T1R domains.

[0178] Receptor-G protein interactions can also be examined. Forexample, binding of the G protein to the receptor, or its release fromthe receptor can be examined. More particularly, in the absence of GTP,an activator will lead to the formation of a tight complex of a Gprotein (all three subunits) with the receptor. This complex can bedetected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors, e.g., by adding an activator to thereceptor and G protein in the absence of GTP, which form a tightcomplex, and then screen for inhibitors by looking at dissociation ofthe receptor-G protein complex. In the presence of GTP, release of thealpha subunit of the G protein from the other two G protein subunitsserves as a criterion of activation. An activated or inhibited G proteinwill in turn alter the properties of target enzymes, channels, and othereffector proteins.

[0179] In another embodiment of the invention, a GTPγS assay may beused. As described above, upon activation of a GPCR, the Gα subunit ofthe G protein complex is stimulated to exchange bound GDP for GTP.Ligand-mediated stimulation of G protein exchange activity can bemeasured in a biochemical assay measuring the binding of addedradioactively-labeled GTPγ³⁵S to the G protein in the presence of aputative ligand. Typically, membranes containing the chemosensoryreceptor of interest are mixed with a complex of G proteins. Potentialinhibitors and/or activators and GTPγS are added to the assay, andbinding of GTPγS to the G protein is measured. Binding can be measuredby liquid scintillation counting or by any other means known in the art,including scintillation proximity assays (SPA). In other assays formats,fluorescently-labeled GTPγS can be utilized.

[0180] 2. Fluorescence Polarization Assays

[0181] In another embodiment, Fluorescence Polarization (“FP”) basedassays may be used to detect and monitor ligand binding. Fluorescencepolarization is a versatile laboratory technique for measuringequilibrium binding, nucleic acid hybridization, and enzymatic activity.Fluorescence polarization assays are homogeneous in that they do notrequire a separation step such as centrifugation, filtration,chromatography, precipitation, or electrophoresis. These assays are donein real time, directly in solution and do not require an immobilizedphase. Polarization values can be measured repeatedly and after theaddition of reagents since measuring the polarization is rapid and doesnot destroy the sample. Generally, this technique can be used to measurepolarization values of fluorophores from low picomolar to micromolarlevels. This section describes how fluorescence polarization can be usedin a simple and quantitative way to measure the binding of ligands tothe T1R polypeptides of the invention.

[0182] When a fluorescently labeled molecule is excited with planepolarized light, it emits light that has a degree of polarization thatis inversely proportional to its molecular rotation. Large fluorescentlylabeled molecules remain relatively stationary during the excited state(4 nanoseconds in the case of fluorescein) and the polarization of thelight remains relatively constant between excitation and emission. Smallfluorescently labeled molecules rotate rapidly during the excited stateand the polarization changes significantly between excitation andemission. Therefore, small molecules have low polarization values andlarge molecules have high polarization values. For example, asingle-stranded fluorescein-labeled oligonucleotide has a relatively lowpolarization value but when it is hybridized to a complementary strand,it has a higher polarization value. When using FP to detect and monitortastant-binding which may activate or inhibit the chemosensory receptorsof the invention, fluorescence-labeled tastants or auto-fluorescenttastants may be used.

[0183] Fluorescence polarization (P) is defined as:$P = \frac{{Int}_{\coprod} - {Int}_{\bot}}{{Int}_{\coprod} + {Int}_{\bot}}$

[0184] Where Π is the intensity of the emission light parallel to theexcitation light plane and Int ⊥ is the intensity of the emission lightperpendicular to the excitation light plane. P, being a ratio of lightintensities, is a dimensionless number. For example, the Beacon® andBeacon 2000™ System may be used in connection with these assays. Suchsystems typically express polarization in millipolarization units (1Polarization Unit=1000 mP Units).

[0185] The relationship between molecular rotation and size is describedby the Perrin equation and the reader is referred to Jolley, M. E.(1991) in Journal of Analytical Toxicology, pp. 236-240, which gives athorough explanation of this equation. Summarily, the Perrin equationstates that polarization is directly proportional to the rotationalrelaxation time, the time that it takes a molecule to rotate through anangle of approximately 68.5° Rotational relaxation time is related toviscosity (η), absolute temperature (T), molecular volume (V), and thegas constant (R) by the following equation:${RotationalRelaxationTime} = \frac{3\quad \eta \quad V}{R\quad T}$

[0186] The rotational relaxation time is small (≈1 nanosecond) for smallmolecules (e.g. fluorescein) and large (≈100 nanoseconds) for largemolecules (e.g. immunoglobulins). If viscosity and temperature are heldconstant, rotational relaxation time, and therefore polarization, isdirectly related to the molecular volume. Changes in molecular volumemay be due to interactions with other molecules, dissociation,polymerization, degradation, hybridization, or conformational changes ofthe fluorescently labeled molecule. For example, fluorescencepolarization has been used to measure enzymatic cleavage of largefluorescein labeled polymers by proteases, DNases, and RNases. It alsohas been used to measure equilibrium binding for protein/proteininteractions, antibody/antigen binding, and protein/DNA binding.

[0187] 3. Solid State and Soluble High Throughput Assays

[0188] In yet another embodiment, the invention provides soluble assaysusing a T1R polypeptide; or a cell or tissue expressing an T1Rpolypeptide. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where the T1Rpolypeptide, or cell or tissue expressing the T1R polypeptide isattached to a solid phase substrate.

[0189] In the high throughput assays of the invention, it is possible toscreen up to several thousand different modulators or ligands in asingle day. In particular, each well of a microtiter plate can be usedto run a separate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 1000 to about 1500different compounds. It is also possible to assay multiple compounds ineach plate well. It is possible to assay several different plates perday; assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention. More recently,microfluidic approaches to reagent manipulation have been developed.

[0190] The molecule of interest can be bound to the solid statecomponent, directly or indirectly, via covalent or non-covalent linkage,e.g., via a tag. The tag can be any of a variety of components. Ingeneral, a molecule which binds the tag (a tag binder) is fixed to asolid support, and the tagged molecule of interest (e.g., the tastetransduction molecule of interest) is attached to the solid support byinteraction of the tag and the tag binder.

[0191] A number of tags and tag binders can be used, based upon knownmolecular interactions well described in the literature. For example,where a tag has a natural binder, for example, biotin, protein A, orprotein G, it can be used in conjunction with appropriate tag binders(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,etc.). Antibodies to molecules with natural binders such as biotin arealso widely available and appropriate tag binders (see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

[0192] Similarly, any haptenic or antigenic compound can be used incombination with an appropriate antibody to form a tag/tag binder pair.Thousands of specific antibodies are commercially available and manyadditional antibodies are described in the literature. For example, inone common configuration, the tag is a first antibody and the tag binderis a second antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

[0193] Synthetic polymers, such as polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates can also form anappropriate tag or tag binder. Many other tag/tag binder pairs are alsouseful in assay systems described herein, as would be apparent to one ofskill upon review of this disclosure.

[0194] Common linkers such as peptides, polyethers, and the like canalso serve as tags, and include polypeptide sequences, such as poly glysequences of between about 5 and 200 amino acids. Such flexible linkersare known to persons of skill in the art. For example, poly(ethelyneglycol) linkers are available from Shearwater Polymers, Inc. Huntsville,Ala. These linkers optionally have amide linkages, sulfhydryl linkages,or heterofunctional linkages.

[0195] Tag binders are fixed to solid substrates using any of a varietyof methods currently available. Solid substrates are commonlyderivatized or functionalized by exposing all or a portion of thesubstrate to a chemical reagent which fixes a chemical group to thesurface which is reactive with a portion of the tag binder. For example,groups which are suitable for attachment to a longer chain portion wouldinclude amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanesand hydroxyalkylsilanes can be used to functionalize a variety ofsurfaces, such as glass surfaces. The construction of such solid phasebiopolymer arrays is well described in the literature. See, e.g.,Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963) (describing solidphase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.,102:259-274 (1987) (describing synthesis of solid phase components onpins); Frank & Doring, Tetrahedron, 44:60316040 (1988) (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry,39(4):718-719 (1993); and Kozal et al., Nature Medicine, 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

[0196] 4. Computer-based Assays

[0197] Yet another assay for compounds that modulate T1R polypeptideactivity involves computer assisted compound design, in which a computersystem is used to generate a three-dimensional structure of an T1Rpolypeptide based on the structural information encoded by its aminoacid sequence. The input amino acid sequence interacts directly andactively with a preestablished algorithm in a computer program to yieldsecondary, tertiary, and quaternary structural models of the protein.The models of the protein structure are then examined to identifyregions of the structure that have the ability to bind, e.g., ligands.These regions are then used to identify ligands that bind to theprotein.

[0198] The three-dimensional structural model of the protein isgenerated by entering protein amino acid sequences of at least 10 aminoacid residues or corresponding nucleic acid sequences encoding a T1Rpolypeptide into the computer system. The nucleotide sequence encodingthe T1R polypeptide, or the amino acid sequence thereof, can be anysequence disclosed herein, and conservatively modified versions thereof.

[0199] The amino acid sequence represents the primary sequence orsubsequence of the protein, which encodes the structural information ofthe protein. At least 10 residues of the amino acid sequence (or anucleotide sequence encoding 10 amino acids) are entered into thecomputer system from computer keyboards, computer readable substratesthat include, but are not limited to, electronic storage media (e.g.,magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,CD ROM), information distributed by internet sites, and by RAM. Thethree-dimensional structural model of the protein is then generated bythe interaction of the amino acid sequence and the computer system,using software known to those of skill in the art.

[0200] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the protein of interest. The software looks atcertain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as “energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

[0201] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0202] Once the structure has been generated, potential ligand-bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of the T1R polypeptide to identify ligands that bind tothe protein. Binding affinity between the protein and ligands isdetermined using energy terms to determine which ligands have anenhanced probability of binding to the protein.

[0203] Computer systems are also used to screen for mutations,polymorphic variants, alleles, and interspecies homologs of T1R genes.Such mutations can be associated with disease states or genetic traits.As described above, GeneChip™ and related technology can also be used toscreen for mutations, polymorphic variants, alleles, and interspecieshomologs. Once the variants are identified, diagnostic assays can beused to identify patients having such mutated genes. Identification ofthe mutated T1R genes involves receiving input of a first nucleic acidor amino acid sequence of a T1R gene, or conservatively modifiedversions thereof. The sequence is entered into the computer system asdescribed above. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in various T1R genes, and mutationsassociated with disease states and genetic traits.

[0204] 5. Cell-based Binding Assays

[0205] In one embodiment, a T1R protein or polypeptide is expressed in aeukaryotic cell as a chimeric receptor with a heterologous, chaperonesequence that facilitates its maturation and targeting through thesecretory pathway. Such chimeric T1R polypeptides can be expressed inany eukaryotic cell, such as HEK-293 cells. Preferably, the cellscomprise a functional G protein, e.g., Gαl5, that is capable of couplingthe chimeric receptor to an intracellular signaling pathway or to asignaling protein such as phospholipase C. Activation of such chimericreceptors in such cells can be detected using any standard method, suchas by detecting changes in intracellular calcium by detecting FURA-2dependent fluorescence in the cell.

[0206] Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor (and possibly othersites as well). Thus, activators will promote the transfer of 32P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature,10:349:117-27 (1991); Bourne et al., Nature, 348:125-32 (1990); Pitcheret al., Annu. Rev. Biochem., 67:653-92 (1998).

[0207] T1R modulation may be assayed by comparing the response of a T1Rpolypeptide treated with a putative T1R modulator to the response of anuntreated control sample. Such putative T1R modulators can includetastants that either inhibit or activate T1R polypeptide activity. Inone embodiment, control samples (untreated with activators orinhibitors) are assigned a relative T1R activity value of 100.Inhibition of a T1R polypeptide is achieved when the T1R activity valuerelative to the control is about 90%, optionally 50%, optionally 25-0%.Activation of a T1R polypeptide is achieved when the T1R activity valuerelative to the control is 110%, optionally 150%, 200-500%, or1000-2000%.

[0208] Changes in ion flux may be assessed by determining changes inionic polarization (i.e., electrical potential) of the cell or membraneexpressing a T1R polypeptide. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques(see, e.g., the “cell-attached” mode, the “inside-out” mode, and the“whole cell” mode, e.g., Ackerman et al., New Engl. J Med.,336:1575-1595 (1997)). Whole cell currents are conveniently determinedusing the standard. Other known assays include: radiolabeled ion fluxassays and fluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol., 88:67-75 (1988); Gonzales& Tsien, Chem. Biol., 4:269277 (1997); Daniel et al., J. Pharmacol.Meth., 25:185-193 (1991); Holevinsky et al., J. Membrane Biology,137:59-70 (1994)). Generally, the compounds to be tested are present inthe range from 1 pM to 100 mM.

[0209] The effects of the test compounds upon the function of thepolypeptides can be measured by examining any of the parametersdescribed above. Any suitable physiological change that affects GPCRactivity can be used to assess the influence of a test compound on thepolypeptides of this invention. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as Ca²⁺,IP3, cGMP, or cAMP.

[0210] Preferred assays for GPCRs include cells that are loaded with ionor voltage sensitive dyes to report receptor activity. Assays fordetermining activity of such receptors can also use known agonists andantagonists for other G protein-coupled receptors as negative orpositive controls to assess activity of tested compounds. In assays foridentifying modulatory compounds (e.g., agonists, antagonists), changesin the level of ions in the cytoplasm or membrane voltage will bemonitored using an ion sensitive or membrane voltage fluorescentindicator; respectively. Among the ion-sensitive indicators and voltageprobes that may be employed are those disclosed in the Molecular Probes1997 Catalog. For G protein-coupled receptors, promiscuous G proteinssuch as Gαl5 and Gαl6 can be used in the assay of choice (Wilkie et al.,Proc. Nat'l Acad. Sci., 88:10049-10053 (1991)). Such promiscuous Gproteins allow coupling of a wide range of receptors.

[0211] Receptor activation typically initiates subsequent intracellularevents, e.g., increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some Gprotein-coupled receptors stimulates the formation of inositoltriphosphate (IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature, 312:315-21 (1984)). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G protein-coupledreceptor function. Cells expressing such G protein-coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

[0212] Other assays can involve determining the activity of receptorswhich, when activated, result in a change in the level of intracellularcyclic nucleotides, e.g., cAMP or cGMP, by activating or inhibitingenzymes such as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Nat'l Acad. Sci.,88:9868-9872 (1991) and Dhallan et al., Nature, 347:184-187 (1990)). Incases where activation of the receptor results in a decrease in cyclicnucleotide levels, it may be preferable to expose the cells to agentsthat increase intracellular cyclic nucleotide levels, e.g., forskolin,prior to adding a receptor-activating compound to the cells in theassay. Cells for this type of assay can be made by co-transfection of ahost cell with DNA encoding a cyclic nucleotide-crated ion channel, GPCRphosphatase and DNA encoding a receptor (e.g., certain glutamatereceptors, muscarinic acetylcholine receptors, dopamine receptors,serotonin receptors, and the like), which, when activated, causes achange in cyclic nucleotide levels in the cytoplasm.

[0213] In a preferred embodiment, T1R polypeptide activity is measuredby expressing a T1R gene in a heterologous cell with a promiscuous Gprotein that links the receptor to a phospholipase C signal transductionpathway (see Offermanns & Simon, J. Biol. Chem., 270:15175-15180(1995)). Optionally the cell line is HEK-293 (which does not naturallyexpress T1R genes) and the promiscuous G protein is Gαl5 (Offermanns &Simon, supra). Modulation of taste transduction is assayed by measuringchanges in intracellular Ca²⁺ levels, which change in response tomodulation of the T1R signal transduction pathway via administration ofa molecule that associates with a T1R polypeptide. Changes in Ca²⁺levels are optionally measured using fluorescent Ca²⁺ indicator dyes andfluorometric imaging.

[0214] In one embodiment, the changes in intracellular cAMP or cGMP canbe measured using immunoassays. The method described in Offermanns &Simon, J. Bio. Chem., 270:15175-15180 (1995), may be used to determinethe level of cAMP. Also, the method described in Felley-Bosco et al.,Am. J. Resp. Cell and Mol. Biol., 11:159-164 (1994), may be used todetermine the level of cGMP. Further, an assay kit for measuring cAMPand/or cGMP is described in U.S. Pat. No. 4,115,538, herein incorporatedby reference.

[0215] In another embodiment, phosphatidyl inositol (PI) hydrolysis canbe analyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with3H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist, to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist, to cpm in the presence of buffer control(which may or may not contain an agonist).

[0216] In another embodiment, transcription levels can be measured toassess the effects of a test compound on signal transduction. A hostcell containing a T1R polypeptide of interest is contacted with a testcompound for a sufficient time to effect any interactions, and then thelevel of gene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by running a timecourse and measuring the level of transcription as a function of time.The amount of transcription may be measured by using any method known tothose of skill in the art to be suitable. For example, mRNA expressionof the protein of interest may be detected using northern blots or theirpolypeptide products may be identified using immunoassays.Alternatively, transcription based assays using reporter gene may beused as described in U.S. Pat. No. 5,436,128, herein incorporated byreference. The reporter genes can be, e.g., chloramphenicolacetyltransferase, luciferase, ′3-galactosidase and alkalinephosphatase. Furthermore, the protein of interest can be used as anindirect reporter via attachment to a second reporter such as greenfluorescent protein (see, e.g., Mistili & Spector, Nature Biotechnology,15:961-964 (1997)).

[0217] The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the T1R polypeptide of interest.A substantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the T1R polypeptide of interest.

[0218] 6. Transgenic Non-human Animals Expressing Chemosensory Receptors

[0219] Non-human animals expressing one or more chemosensory receptorsequences of the invention, can also be used for receptor assays. Suchexpression can be used to determine whether a test compound specificallybinds to a mammalian taste transmembrane receptor polypeptide in vivo bycontacting a non-human animal stably or transiently transfected with anucleic acid encoding a chemosensory receptor or ligand-binding regionthereof with a test compound and determining whether the animal reactsto the test compound by specifically binding to the receptorpolypeptide.

[0220] Animals transfected or infected with the vectors of the inventionare particularly useful for assays to identify and characterizetastants/ligands that can bind to a specific or sets of receptors. Suchvector-infected animals expressing human chemosensory receptor sequencescan be used for in vivo screening of tastants and their effect on, e.g.,cell physiology (e.g., on taste neurons), on the CNS, or behavior.

[0221] Means to infect/express the nucleic acids and vectors, eitherindividually or as libraries, are well known in the art. A variety ofindividual cell, organ, or whole animal parameters can be measured by avariety of means. The T1R sequences of the invention can be for exampleexpressed in animal taste tissues by delivery with an infecting agent,e.g., adenovirus expression vector.

[0222] The endogenous chemosensory receptor genes can remain functionaland wild-type (native) activity can still be present. In othersituations, where it is desirable that all chemosensory receptoractivity is by the introduced exogenous hybrid receptor, use of aknockout line is preferred. Methods for the construction of non-humantransgenic animals, particularly transgenic mice, and the selection andpreparation of recombinant constructs for generating transformed cellsare well known in the art.

[0223] Construction of a “knockout” cell and animal is based on thepremise that the level of expression of a particular gene in a mammaliancell can be decreased or completely abrogated by introducing into thegenome a new DNA sequence that serves to interrupt some portion of theDNA sequence of the gene to be suppressed. Also, “gene trap insertion”can be used to disrupt a host gene, and mouse embryonic stem (ES) cellscan be used to produce knockout transgenic animals (see, e.g., Holzschu,Transgenic Res 6:97-106 (1997)). The insertion of the exogenous istypically by homologous recombination between complementary nucleic acidsequences. The exogenous sequence is some portion of the target gene tobe modified, such as exonic, intronic or transcriptional regulatorysequences, or any genomic sequence which is able to affect the level ofthe target gene's expression; or a combination thereof. Gene targetingvia homologous recombination in pluripotential embryonic stem cellsallows one to modify precisely the genomic sequence of interest. Anytechnique can be used to create, screen for, propagate, a knockoutanimal, e.g., see Bijvoet, Hum. Mol. Genet. 7:53-62 (1998); Moreadith,J. Mol. Med. 75:208-216 (1997); Tojo, Cytotechnology 19:161-165 (1995);Mudgett, Methods Mol. Biol. 48:167-184 (1995); Longo, Transgenic Res.6:321-328 (1997); U.S. Pat. Nos. 5,616,491; 5,464,764; 5,631,153;5,487,992; 5,627,059; 5,272,071; WO 91/09955; WO93/09222; WO 96/29411;WO 95/31560; WO 91/12650.

[0224] The nucleic acids of the invention can also be used as reagentsto produce “knockout” human cells and their progeny. Likewise, thenucleic acids of the invention can also be used as reagents to produce“knock-ins” in mice. The human or rat T1R gene sequences can replace theorthologous T1R in the mouse genome. In this way, a mouse expressing ahuman or rat T1R is produced. This mouse can then be used to analyze thefunction of human or rat T1Rs, and to identify ligands for such T1Rs.

F. Modulators

[0225] The compounds tested as modulators of a T1R family member can beany small chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a T1R gene. Typically, test compoundswill be small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds can be dissolved in aqueousor organic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) and the like.

[0226] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0227] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0228] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493(1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci., 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara etal., J. Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,114:9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),oligocarbamates (Cho et al., Science, 261:1303 (1993)), peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleicacid libraries (Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) andPCT/US96/10287), carbohydrate libraries (Liang et al., Science,274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (benzodiazepines, Baum, C&EN, January 18, page 33(1993); thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pynrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

[0229] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS (Advanced Chem Tech,Louisville Ky.), Symphony (Rainin, Woburn, Mass.), 433A (AppliedBiosystems, Foster City, Calif.), 9050 Plus (Millipore, Bedford,Mass.)). In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Tripos,Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; MartekBiosciences; Columbia, Md.; etc.).

[0230] In one aspect of the invention, the T1R modulators can be used inany food product, confectionery, pharmaceutical composition, oringredient thereof to thereby modulate the taste of the product,composition, or ingredient in a desired manner. For instance, T1Rmodulators which enhance sweet taste sensation can be added to sweeten aproduct or composition, while T1R modulators which block undesirabletaste sensations can be added to improve the taste of a product orcomposition.

G. Methods for Representing and Predicting the Perception of Taste

[0231] The invention also preferably provides methods for representingthe perception of taste and/or for predicting the perception of taste ina mammal, including in a human. Preferably, such methods may beperformed by using the receptors and genes encoding said T1Rpolypeptides disclosed herein.

[0232] Also contemplated as within the invention, is a method ofscreening one or more compounds for the presence of a taste detectableby a mammal, comprising: contacting said one or more compounds with thedisclosed receptors, preferably wherein the mammal is a human. Alsocontemplated as within the invention, is a method for representing tasteperception of a particular taste in a mammal, comprising the steps of:providing values X₁ to X_(n) representative of the quantitativestimulation of each of n chemosensory receptors of said vertebrate,where n is greater than or equal to 2; and generating from said values aquantitative representation of taste perception. The chemosensoryreceptors may be a chemosensory receptor disclosed herein, therepresentation may constitutes a point or a volume in n-dimensionalspace, may constitutes a graph or a spectrum, and may constitutes amatrix of quantitative representations. Also, the providing step maycomprise contacting a plurality of recombinantly-produced chemosensoryreceptors with a test composition and quantitatively measuring theinteraction of said composition with said receptors.

[0233] Also contemplated as within the invention, is a method forpredicting the taste perception in a mammal generated by one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal, comprising the steps of: providing values X₁ to X_(n)representative of the quantitative stimulation of each of n chemosensoryreceptors of said vertebrate, where n is greater than or equal to 2 forone or more molecules or combinations of molecules yielding known tasteperception in a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding known taste perceptionin a mammal, providing values X₁ to X_(n) representative of thequantitative stimulation of each of n chemosensory receptors of saidvertebrate, where n is greater than or equal to 2, for one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal; and generating from said values a quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal, and predicting the taste perception in a mammal generatedby one or more molecules or combinations of molecules yielding unknowntaste perception in a mammal by comparing the quantitativerepresentation of taste perception in a mammal for the one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal to the quantitative representation of taste perception in amammal for the one or more molecules or combinations of moleculesyielding known taste perception in a mammal. The chemosensory receptorsused in this method may include a chemosensory receptor disclosedherein.

[0234] In another embodiment, novel molecules or combinations ofmolecules are generated which elicit a predetermined taste perception ina mammal by determining a value of taste perception in a mammal for aknown molecule or combinations of molecules as described above;determining a value of taste perception in a mammal for one or moreunknown molecules or combinations of molecules as described above;comparing the value of taste perception in a mammal for one or moreunknown compositions to the value of taste perception in a mammal forone or more known compositions; selecting a molecule or combination ofmolecules that elicits a predetermined taste perception in a mammal; andcombining two or more unknown molecules or combinations of molecules toform a molecule or combination of molecules that elicits a predeterminedtaste perception in a mammal. The combining step yields a singlemolecule or a combination of molecules that elicits a predeterminedtaste perception in a mammal.

[0235] In another embodiment of the invention, there is provided amethod for simulating a taste, comprising the steps of: for each of aplurality of cloned chemosensory receptors, preferably human receptors,ascertaining the extent to which the receptor interacts with thetastant; and combining a plurality of compounds, each having apreviously-ascertained interaction with one or more of the receptors, inamounts that together provide a receptor-stimulation profile that mimicsthe profile for the tastant. Interaction of a tastant with achemosensory receptor can be determined using any of the binding orreporter assays described herein. The plurality of compounds may then becombined to form a mixture. If desired, one or more of the plurality ofthe compounds can be combined covalently. The combined compoundssubstantially stimulate at least 75%, 80%, or 90% of the receptors thatare substantially stimulated by the tastant.

[0236] In another preferred embodiment of the invention, a plurality ofstandard compounds are tested against a plurality of chemosensoryreceptors to ascertain the extent to which the receptors each interactwith each standard compound, thereby generating a receptor stimulationprofile for each standard compound. These receptor stimulation profilesmay then be stored in a relational database on a data storage medium.The method may further comprise providing a desired receptor-stimulationprofile for a taste; comparing the desired receptor stimulation profileto the relational database; and ascertaining one or more combinations ofstandard compounds that most closely match the desiredreceptor-stimulation profile. The method may further comprise combiningstandard compounds in one or more of the ascertained combinations tosimulate the taste.

H. Kits

[0237] T1R genes and their homologs are useful tools for identifyingchemosensory receptor cells, for forensics and paternity determinations,and for examining taste transduction. T1R family member-specificreagents that specifically hybridize to T1R nucleic acids, such as T1Rprobes and primers, and T1R specific reagents that specifically bind toa T1R polypeptide, e.g., T1R antibodies are used to examine taste cellexpression and taste transduction regulation.

[0238] Nucleic acid assays for the presence of DNA and RNA for a T1Rfamily member in a sample include numerous techniques are known to thoseskilled in the art, such as southern analysis, northern analysis, dotblots, RNase protection, S1 analysis, amplification techniques such asPCR, and in situ hybridization. In in situ hybridization, for example,the target nucleic acid is liberated from its cellular surroundings insuch as to be available for hybridization within the cell whilepreserving the cellular morphology for subsequent interpretation andanalysis. The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques, 4:230250 (1986); Haaseet al., Methods in Virology, vol. VII, pp. 189-226 (1984); and NucleicAcid Hybridization: A Practical Approach (Names et al., eds. 1987). Inaddition, a T1R polypeptide can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing a recombinant T1Rpolypeptide) and a negative control.

[0239] The present invention also provides for kits for screening formodulators of T1R family members. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: T1R nucleic acids orproteins, reaction tubes, and instructions for testing T1R activity.Optionally, the kit contains a biologically active T1R receptor. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user.

EXAMPLES

[0240] In the protein sequences presented herein, the one-letter code Xor Xaa refers to any of the twenty common amino acid residues. In theDNA sequences presented herein, the one letter codes N or n refers toany of the of the four common nucleotide bases, A, T, C, or G.

[0241] Example 1—hT1R3

[0242] The hT1R3 genomic DNA is provided below as SEQ ID NO 1 and SEQ IDNO 2 with predicted coding sequences (cds) shown in boldface. The breakbetween the 5′ and 3′ contigs is shown as elipses (‘. . . ’). The hT1R3predicted cds are described in SEQ ID NO 3. Finally, a preferred,predicted hT1R3 amino acid sequence is provided as SEQ ID NO 4, usingthe one-letter code for the amino acids.

[0243] hT1R3 Genomic DNA—5′ contig (SEQ ID NO 1)AGCCTGGCAGTGGCCTCAGGCAGAGTCTGACGCGCACAAACTTTCAGGCCCAGGAAGCGA (SEQ IDNO: 1) GGACACCACTGGGGCCCCAGGGTGTGGCAAGTGAGGATGGCAAGGGTTTTGCTAAACAAATCCTCTGCCCGCTCCCCGCCCCGGGCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTACAGAGGTGGGACGGCCTGGGTCGGGGTCAGGGTGACCAGGTCTGGGGTGCTCCTGAGCTGGGGCCGAGGTGGCCATCTGCGGTTCTGTGTGGCCCCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGTGGGGCGCCCCCCACCATCACCCACCCCCAACCAACCCCTGCCCCGTGGGAGCCCCTTGTGTCAGGAGAATGC

[0244] hT1R3 Genomic DNA—3′ contig (SEQ ID NO 2) . . .TACATGCACCCCACCCAGCCCTGCCCTGGGAGCCCTGTGTCAGAAGATGC (SEQ ID NO 2)TCTTGGCCTTGCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGGTGAGCCCGGGAGATGGGGGTGTGCTGTCCTCTGCATGTGCCCAGGCCACCAGGCACGGCCACCACGCCTGAGCTGGAGGTGGCTGGCGGCTCAGCCCCGTCCCCCGCCCGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGGTGAGGTGAGGGTGGGTGTGCCAGGCGTGCCCGTGGTAGCCCCCGCGGCAGGGCGCAGCCTGGGGGTGGGGGCCGTTCCAGTCTCCCGTGGGCATGCCCAGCCGAGCAGAGCCAGACCCCAGGCCTGTGCGCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGGTGAGCCGCCTTCCCGGCAGGCGGGGGTGGGAACGCAGCAGGGGAGGGTCCTGCCAAGTCCTGACTCTGAGACCAGAGCCCACAGGGTACAAGACGAACACCCAGCGCCCTTCTCCTCTCTCACAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATCTCAGCCCCGGTGAACCCAGACTTAGCTGCGATCCCCCCCAAGCCAGCAATGACCCGTGTCTCGCTACAGAGACCCTCCCGCTCTAGGTTCTGACCCCAGGTTGTCTCCTGACCCTGACCCCACAGTGAGCCCTAGGCCTGGAGCACGTGGACACCCCTGTGACCATC

[0245] hT1R3 Full-length Genomic DNA (SEQ ID NO 20)AGCCTGGCAGTGGCCTCAGGCAGAGTCTGACGCGCACAAACTTTCAGGCCCAGGAAGCGA (SEQ ID NO20) GGACACCACTGGGGCCCCAGGGTGTGGCAAGTGAGGATGGCAAGGGTTTTGCTAAACAAATCCTCTGCCCGCTCCCCGCCCCGGGCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTACAGAGGTGGGACGGCCTGGGTCGGGGTCAGGGTGACCAGGTCTGGGGTGCTCCTGAGCTGGGGCCGAGGTGGCCATCTGCGGTTCTGTGTGGCCCCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGTGGGGCGCCCCCCACCATCACCCACCCCCAACCAACCCCTGCCCCGTGGGAGCCCCTTGTGTCAGGAGAATGCTACATGCACCCCACCCAGCCCTGCCCTGGGAGCCCTGTGTCAGAAGATGCTCTTGGCCTTGCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTCCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGGTGAGCCCGGGAGATGGGGGTGTGCTGTCCTCTGCATGTGCCCAGGCCACCAGGCACGGCCACCACGCCTGAGCTGGAGGTGGCTGGCGGCTCAGCCCCGTCCCCCGCCCGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGGTGAGGTGAGGGTGGGTGTGCCAGGCGTGCCCGTGGTAGCCCCCGCGGCAGGGCGCAGCCTGGGGGTGGGGGCCGTTCCAGTCTCCCGTGGGCATGCCCAGCCGAGCAGAGCCAGACCCCAGGCCTGTGCGCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGGTGAGCCGCCTTCCCGGCAGGCGGGGGTGGGAACGCAGCAGGGGAGGGTCCTGCCAAGTCCTGACTCTGAGACCAGAGCCCACAGGGTACAAGACGAACACCCAGCGCCCTTCTCCTCTCTCACAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTCACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATCTCAGCCCCGGTGAACCCAGACTTAGCTGCGATCCCCCCCAAGCCAGCAATGACCCGTGTCTCGCTACAGAGACCCTCCCGCTCTAGGTTCTGACCCCAGGTTGTCTCCTGACCCTGACCCCACAGTGAGCCCTAGGCCTGGAGCACGTGGACACCCCTGTGACCATC

[0246] hT1R3 Predicted cds (SEQ ID NO 3)ATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGG (SEQ ID NO3) CCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCACTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCGTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGACGACATCGCCTGCACCTTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACA TGAGTGA

[0247] hT1R3 Conceptual Translation (SEQ ID NO 4)MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYVLGGLFPLGEAEEAGLRSRTRPSSPVCT (SEQ IDNO 4) RFSSNGLLWALAMKMAVEEINNKSDLLPGLRLGYDLFDTCSEPVVAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHSSELAMVTGKFFSFFLMPHYGASMELLSARETFPSFFRTVPSDRVQLTAAAELLQEFGWNWVAALGSDDEYGRQGLSIFSALAAARGICIAHEGLVPLPRADDSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWVASEAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALATDPAFCSALGEREQGLEEDVVGQRCPQCDCITLQNVSAGLNHHQTFSVYAAVYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRFDSSGNVDMEYDLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDNQKPVSRCSRQGQEGQVRRVKGFHSCCYDCVDCEAGSYRQNPDDIACTFCGQDEWSPERSTRCFRRRSRFLAWGEPAVLLLLLLLSLALGLVLAALGLFVHHRDSPLVQASGGPLACFGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHLPLTGCLSTLFLQAAEIFVESELPLSWADRLSGCLRGPWAWLVVLLAMLVEVALGTWYLVAFPPEVVTDWHMLPTEALVHCRTRSWVSFGLAHATNATLAFLCFLGTFLVRSQPGCYNRARGLTFAMLAYFITWVSFVPLLANVQVVLRPAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLGGGPGDAQGQNDGNTGNQGKHE

Example 2—rT1R3 and mT1R3

[0248] Segments of the rat and mouse T1R3 genes were isolated by PCRamplification from genomic DNA using degenerate primers based on thehuman T1R3 sequence. The degenerate primers SAP077(5′-CGNTTYYTNGCNTGGGGNGARCC-3′; SEQ ID NO 5) and SAP079(5′-CGNGCNCGRTTRTARCANCCNGG-3′; SEQ ID NO 6) are complementary to humanT1R3 residues RFLAWGEPA (corresponding to SEQ ID NO 7) and PGCYNRAR(corresponding to SEQ ID NO 8), respectively. The PCR products werecloned and sequenced. Plasmid SAV115 carries a cloned segment of themouse T1R3 gene, and SAV118 carries a segment of the rat gene. Thesesequences, shown below, clearly represent the rodent counterparts ofhuman T1R3, since the mouse segment is 74% identical to thecorresponding segment of human T1R3, and the rat segment is 80%identical to the corresponding segment of human T1R3. The mouse and ratsegments are 88% identical. No other database sequences are more than40% identical to these T1R3 segments.

[0249] SAV115 Mouse T1R3 Segment in Sense Orientation (SequenceCorresponding to Degenerate Primer Removed) (SEQ ID NO 9)GTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCT (SEQ IDNO 9) CTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGCGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGACTCTGGCCTGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGACCGCTTCACCAGAAGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAACCTGGGCTTGGTGCACATCACCAATGCAATGGTAGCTTTTCTCTGCTTTCTGGGCACTTTCCTGGTACAAGACCA G

[0250] mT1R3 Segment, Conceptual Translation (SEQ ID NO 10)VLSLLLLLCLVLGLALAALGLSVHHWDSPLVQASGGSQFCFGLICLGLFCLSVLLFPGRPSSASC (SEQID NO 10)LAQQPMAHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRDSGLLVVLLATFVEAALCAWYLTASPEVVTDWSVLPTEVLEHCHVRSWVNLGLVHITNAMVAFLCFLGTFLVQDQ

[0251] SAV118 Rat T1R3 Segment in Sense Orientation (SequenceCorresponding to Degenerate Primer Removed) (SEQ ID NO 11)GTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGACACTGGCTGCCCTGGGGC (SEQ ID NO11) TCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGGTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTTGCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGTGGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTACTGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGGCAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAGGGGTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACA AAGCCAG

[0252] rT1R3 Segment, Conceptual Translation (SEQ ID NO 12)VLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASGGSLFCFGLICLGLFCLSVLLFPGRPRSASC (SEQID NO 12) LAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPWAWLVVLLATLVEAALCAWYLMAFPPEVVTDWQVLPTEVLEHCRMRSWVSLGLVHITNAGVAFLCFLGTFLVQSQ

Example 3—Cloning of rT1R3

[0253] The mT1R3 and rT1R3 fragments identified above as SEQ ID NOs 9and 11 were used to screen a rat taste tissue-derived cDNA library. Onepositive clone was sequenced and found to contain the full-length rT1R3sequence presented below as SEQ ED NO 13. Sequence comparison to themT1R3 and rT1R3 partial sequences and to the full-length hT1R3 sequenceestablished that this cDNA represents the rat counterpart to hT1R3. Forexample, the pairwise amino acid identity between rT1R3 and hT1R3 isapproximately 72%, whereas the most related annotated sequence in publicDNA sequence data banks is only approximately 33% identical to rT1R3.

[0254] rT1R3 Predicted cds (SEQ. ID NO. 13)ATGCCGGGTTTGGCTATCTTGGGCCTCAGTCTGGCTGCTTTCCTGGAGCTTGGGATGGGGT (SEQ ID NO13) CCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTATATATTGGGTGGACTATTTCCCCTGGGCACAACTGAGGAGGCCACTCTCAACCAGAGAACACAGCCCAACGGCATCCTATGTACCAGGTTCTCGCCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTAGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGACTGGGCTATGACCTGTTTGACACATGCTCAGAGCCAGTGGTCACCATGAAGCCCAGCCTCATGTTCATGGCCAAGGTGGGAAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATTGGTCCCCACTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTCATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGGGAAACATTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAGCTGCAGGCCGTTGTGACACTGTTGCAGAATTTCAGCTGGAACTGGGTGGCTGCCTTAGGTAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTTCTGGTCTGGCCAACTCACGAGGTATCTGCATTGCACACGAGGGGCTGGTGCCACAACATGACACTAGTGGCCAACAATTGGGCAAGGTGGTGGATGTGCTACGCCAAGTGAACCAAAGCAAAGTACAGGTGGTGGTGCTGTTTGCATCTGCCCGTGCTGTCTACTCCCTTTTTAGCTACAGCATCCTTCATGACCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCCTGGCTGACCTCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTTCTTGGGTTTCTGCAGCGCGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCGCCTTGCCCTAGCTGCTGACCCAACATTCTGTGCCTCCCTGAAAGCTGAGTTGGATCTGGAGGAGCGCGTGATGGGGCCACGCTGTTCACAATGTGACTACATCATGCTACAGAACCTGTCATCTGGGCTGATGCAGAACCTATCAGCTGGGCAGTTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTGTGGCTCAGGCCCTTCACAACACCCTGCAGTGCAATGTCTCACATTGCCACACATCAGAGCCTGTTCAACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCGTGCTCGAGACTTGACACTGCAGTTTGATGCCAAAGGGAGTGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTACTACATACTGTAGGCACCTTCAACGGCACCCTTCAGCTGCAGCACTCGAAAATGTATTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGCTCCCGGCAGTGCAAAGATGGCCAGGTGCGCAGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGTGTGGACTGCAAGGCAGGGAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTGGCAAGGATCAGTGGTCCCCAGAAAAAAGCACAACCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAGCCAGCTGTGCTGTCACTTCTCCTGCTGCTTTGCCTGGTGCTGGGCCTGACACTGGCTGCCCTGGGGCTCTTTGTCCACTACTGGGACAGCCCTCTTGTTCAGGCCTCAGGTGGGTCACTGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTGAGTGTCCTTCTGTTCCCAGGACGACCACGCTCTGCCAGCTGCCTTGCCCAACAACCAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAGCCGAGATCTTTGTGGAGTCTGAGCTGCCACTGAGTTGGGCAAACTGGCTCTGCAGCTACCTTCGGGGCCCCTGGGCTTGGCTGGTGGTACTGCTGGCCACTCTTGTGGAGGCTGCACTATGTGCCTGGTACTTGATGGCTTTCCCTCCAGAGGTGGTGACAGATTGGCAGGTGCTGCCCACGGAGGTACTGGAACACTGCCGCATGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCACCAATGCAGTGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGTCGCTATAACCGTGCCCGTGGCCTCACCTTCGCCATGCTAGCTTATTTCATCATCTGGGTCTCTTTTGTGCCCCTCCTGGCTAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCTTATTCTGTGCCCTGGGCATCCTGGCCACCTTCCACCTGCCCAAATGCTATGTACTTCTGTGGCTGCCAGAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAGCCCCAAGGAAGCATCAGATGGGAATAGTGGTAGTAGTGAGGCAACTCGGGGACACAGTGAATGA

[0255] rT1R3 Conceptual Translation (SEQ. ID NO. 14)MPGLAILGLSLAAFLELGMGSSLCLSQQFKAQGDYILGGLFPLGTTEEATLNQRTQPNGILCTRF (SEQID NO 14) SPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLFDTCSEPVVTMKPSLMFMAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELALITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLLQNFSWNWVAALGSDDDYGREGLSIFSGLANSRGICIAHEGLVPQHDTSGQQLGKVVDVLRQVNQSKVQVVVLFASARAVYSLFSYSILHDLSPKVWVASESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETRLALAADPTFCASLKAELDLEERVMGPRCSQCDYIMLQNLSSGLMQNLSAGQLHHQIFATYAAVYSVAQALHNTLQCNVSHCHTSEPVQPWQLLENMYNMSFRARDLTLQFDAKGSVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQHSKMYWPGNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTCTPCGKDQWSPEKSTTCLPRRPKFLAWGEPAVLSLLLLLCLVLGLTLAALGLFVHYWDSPLVQASGGSLFCFGLICLGLFCLSVLLFPGRPRSASCLAQQPMAHLPLTGCLSTLFLQAAEIFVESELPLSWANWLCSYLRGPWAWLVVLLATLVEAALCAWYLMAFPPEVVTDWQVLPTEVLEHCRMRSWVSLGLVHITNAVLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFIIWVSFVPLLANVQVAYQPAVQMGAILFCALGILATFHLPKCYVLLWLPELNTQEFFLGRSPKEASDGNSGSSEATRGHSE

Example 4—Expression of mT1R3

[0256] The above described mouse T1R3 fragment contained in SAV115 wasPCR amplified using M13forward and M13reverse primers and then gelpurified. The T1R3 DNA template was placed into an in vitrotranscription labeling reaction where Digoxigenin labeled UTP wasincorporated into an antisense cRNA probe. This probe was hybridized toadult mouse taste tissue containing cicumvallate papillae. The T1R3 insitu hybridization and detection were performed following the protocolof Schaeren-Wiemers et al., Histochemistry, 100:431-400 (1993). Briefly,fresh frozen mouse tongue was sectioned at 14 μm and prepared forhybridization. 200 ng/mL of the antisense Digoxigenin T1R3 probe washybridized for 14 hours at 72° C. Posthybridization consisted of a0.2×SSC wash at 72° C. Digoxigenin detection was accomplished byincubation with 1:5000 dilution of anti-DIG Alkaline Phosphataseantibody followed by a 12-hour reaction of the phosphatase in NBT/BCIP.FIG. 1 shows T1R3 gene expression in taste buds of mouse circumvallatepapillae.

Example 5—hT1R1

[0257] The human ortholog (Database accession no. AL159177) of a rattaste receptor, designated rT1R1, is provided below as SEQ ID NO 15.Predicted cds are indicated in bold and some intronic sequence intervalsare denoted as runs of N. The nucleotide and conceptually-translatedhT1R1 sequences are also described herein as SEQ ID NO 16 and 17,respectively

[0258] hT1R1 Genomic DNA (SEQ ID NO 15)GAGAATCTCGCGAGATCCCGTCGGTCCGCCCCGCTGCCCTCCCAGCTGCCGAAAAGAGGG (SEQ ID NO15) GCCTCCGAGCCGCCGGCGCCCTCTGCCGGCAACCTCCGGAAGCACACTAGGAGGTTCCAGCCGATCTGGTCGAGGGGCTCCACGGAGGACTCCATTTACGTTACGCAAATTCCCTACCCCAGCCGGCCGGAGAGAGAAAGCCAGAAACCTCGCGACCAGCCATGGGCCACCTCTCCGGAAAAACACCGGGATATTTTTTTTCTCCTGCAGAAAAAGCTTTAGGATTGGCAGTTTAAACAAAACATGTCTATTTGCATACCTTCGGTTTGCATGCATTTGTTTCGAAGTGAGCAACCCTGGGTAACAAGGCGAAAGTATATGACAATTTGCTCAGAATCTTAATGTCAGAAAACTGGAGACTGGGGCAGGGGGGTGTCGACTCAAAGCTGTGTCTCATTTAGTAAACTGAGGCCCAGGTAAAAAGTTCTGAAACCTCGCAACACCCGGAGAAATTGTGTTCCAGCCTCCCACCTCGCCCCAAAATGCCAGAGCTCTTTTTCTAAGCCAGGTGAAGTCACAGAGCGTGGACAGAACCCACAACCGTCCAGAGGAAGGGTCACTGGGTGCCACCTGGTTTGCATCTGTGGCCTCGTCCTGCCCAGTTCCTGAGTGGGACCGCAGGCCCGGAATGTCAAGGCAAACAGTCCTGCTTCAGCCACTGGGCTCCAGTCCCACCCCTTTTGGGGGCCTGAAGTTAGGAAGCATCCGGCAGCTGCCTTCTATTTAAGCAACTGGCCTCCTTAGAGGCCACTCCTTGGCCATGCCAGGCGCGGGCATCTGGCCAGCATGCTGCTCTGCACGGCTCGCCTGGTCGGCCTGCAGCTTCTCATTTCCTGCTGCTGGGCCTTTGCCTGCCATAGCACGGAGTCTTCTCCTGACTTCACCCTCCCCGGAGATTACCTCCTGGCAGGCCTGTTCCCTCTCCATTCTGGCTGTCTGCAGGTGAGGCACAGACCCGAGGTGACCCTGTGTGACAGGTGAGTGAGGGGCCAGCAGAGCCACACTTAGTGGGACCCCTGGCTATAGGGCCCCTCTGGCTGCCATCCTCCAAACAGGACCTTGCCTCTGCCTTTGCCCCTTGAACTGTCCCCAGGCCTTGTTCATCAATCCACTTGCCACCTAAGTGCTGGCTAGACCTTCCTAGACACTTCGGCCAGTTTCCAATTATTTCACCCTTGCTGTTAGAATGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAATTCCTTAAACTAAATTTCTCACTTTCTCTCTCTCTCTGGAAAACACTGACTAATGTAGCAGGTTTCTCTGCTCCAGGACTTCAGGACCTTTTCGATGCTAATAAGTTTCTCCATCAGGGCCAGCTTGTTCCTCCTACTGAGCTTGAGAGCCCTTGTTGAAGTTGTGGTTTGGGGGACTGGACCGATGACGTCAAAGGTTCCCTTTGCTCCCAAGCCTCAGAGTCTAGGAGGCCAGAGGGTCTCAGCAGGCCTTTGTCCTTCTCAGCTGTCTCTTACTGGCTTTCTCCACAGGTCTTGTAGCTTCAATGAGCATGGCTACCACCTCTTCCAGGCTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTACCAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCACATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTGCTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCCCATGGTAAGCTGGAGCCTCAGACCTTTGCCCATCTCCCTTCAGGCAAGTCTGGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCACCATGCCCGGCTAATTTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGCTGGTCGCAAACTCCTAACCTCGTGATCCACCCACCTCGGCCTCCCAATGTGCTGGGATTACAGGTGTGAGCCACTGCACCGGGCCATAATGTATTAATATAATAAAATAATTATACAACTCACCATAATGTAGAATCAGTGGGAGCCCTGAGCTTGTTTTCCTACAACTAGATGGTCCCATCTGGGGGTGATGGGAGACAGTGACAGATCATCAGACATTAGATTCTCATAAGTAGCGTGCAACCCAGATCCCTCGCATGTGCAGTTCACAGTAGGGTTCAAGCTCCTACAAGAATCTGATGCTGCTGCTGATCTGACAGGAGGGGAGCAGCTGTAAATACAGATGAAGCTTCGCTTACTCACCAGCTGCTCACCTCCTCCTGTGAGGCCCGGTTCCTAACAGGCCACTGACCTAACTTCTGCCCTGACCTACACATGCTTCTCTTCTTCCTTGCAAACTGCCTCCAGTGGAAGTCCCTGAAGGTCCCCAAACACACGGGACTATTTCACTCCTATGCAGGTTTTGTCTCCTTTGCTTGGAATGCATCCCCTCACCCCTTGTCCCCAGGCAGATTCCCACCCCTCCCCCAGAACCTGCCCCAGTGGAGCCTTCGCAGGTGATTTGTCAGTTTCACAGGCTGAGGGGTGCTCTCCTGGTCTCCCCGGCTCCCTGTATCCCCACACCCAGCACAGGGCCAGGCACTGGGGGGGCCTTCAGTGGAGACTGAAATGGCTGAACGGGACCTCCCATAGATTAGCTATGCGGCCAGCAGCGAGACGCTCAGCGTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCTGCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGGAGAACCAGGCCACTGGTCAGGGGATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAGAGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGCCAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCTCCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCGTGGCCATCCAGAAGAGGGCTGTCCCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGAAGGCCCCTAGGCCTTGCCACAAGGGCTCCTGGTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCATGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCTGGAGCTTGTTCCAGGGGCCGAGTCTACCCCTGGCAGGTAAGAGAGCCCACCGCAGCACCTCCTGTCAGGGAGAACAGCCAATCCTGAGATGAGCAGAGTGGGCACTCTCCGGTGACTCTAAATGCGAAGGGGGATAAATGCCACTAACTTGAGGTTTTTTGTTTTGTTTTGTTTTGTTTTTTGAGACAGTCTGGGTCTGTCACCCAGGCTGCAGTGTAGTGATGCGATCTCGGCTCTCTGCAACTTCCACCTCCTGGGTTCAAGTGATTCTCTTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGCACCCACCACCATGCCTGGATAATTTTTCTTTTCTTTTTTTTTTTTTTGAGATAGAGTCTCGCTGTGTTGCCCAGGCTGGAATGCAGTGGTGCGATCTTGGCTCACTGTGAGCTCCGCCTCCCAGGTTCACTCCATTCCCCTGCCTCAGCCTCCCAAGTAGGTGGGACTACGGGCGCCCGCCACCACGCCCAGCTAATTTTTTTTGTATTTTGAGTAGAGACGGGGTTTCACCATGTTAGCCAGGATGGTCTCAATCTCCTGACCTTGTCATCCGCCCACCTCGTCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCACCCGGCCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGGCATCAAGTGATCCTCCTGCTTCGGCCTCCCAAAGTGCTGGGATTACAGGCATTAGCTCTCTTCTCTTAGACAGATCTTTCTCTCTGATCCTTGCCTTCTCTCACCCACTGTGTCTTGGAAGTGTCAAGTGATAAGATCCAGGGCTAAAACTGTCTGTAAAGGAGTGTTTGTTAGAGGCCTCCTCTCAGGAGGTTGGTGGGGAAGATTGAGGGGCTTCCTAAGAAGGAAGGGACGAGACCTTCCTGATGGGCTGAAACCACCAGGACGGAAACCCAGGAAGGCCCCAGGCCTTGCTTCTGGGACCATGTGGGTCTGTGCTGTCTGTGGTGGCTTCATGATACGCGTTTCTTTCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAAGGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCAAGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGGCACGGAAAGGACAACCAGGTAATGGGGATGTGGCTACTCACCATGTAACTGGCTTATGGGCAACCTAGAGCCTGGGGGTGATGCTGACACAGTGTACAGGGAGCAGGAGGGGGGCCCCAGGGGTCCAGCTGGCACCACTCTACCCATCCTGGCCAGGGAAGCAGGGAAGACACTCCGTAGGCGAGTGTGCAGATGCCCTGGGGCGGAAGTTCACACGACCAGGGGCCCTGCCCTGGGAGTGAGCCCTGAGGGCAGATGCACAGAGATTCTGTTTTCTGTTCCACATGTGAGCTGTCCTTTGACTTGGGCCCCTACGTGTGGCCCCTCTGGCTTCTTACAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGGTTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGTGGTGAGTGGGCAATGGAGCAGGCGAGCTACCCAGCACTCCCGGGGGCTGCACGGTGGAGGGAGGGCCTCCCTTGGGCCCCATGTGCCGTGCCCCAGAACCAAGGCCCAGTCACTGGGCTGCCAGTTAGCTTCAGGTTGGAGGACACCTGCTACCAGACAGAATTCTGATCAAGAGAATCAGCCACTGGGTGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTGGATCACTTGAGGTCGGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACCAAAAATATAAAAAATTAGCTGGGTGTGGTGGCGCGTGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCACTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCCAAGATGCATTCCAGCCTGGACCACAAAGCGAGAATTCGTCCCCCCAAAAAAAGAAAGGAGGCCGGGCGCGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAAAAGTTAGCCGGGCGTTGTGGCGTGTGCCTGTAATTCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGCACCATTGCACTCCAGCCTGGGCGACAAGAGAAAAACTCTGTCTCAAAAAAAAAGAAAGAAAGAAAGAATTAGCCAACTGAAAGCCTTAGACTGAGGTGTGTCCTCTGTTAGAGAGCTGTCATCACAACTCCTACAAAAGCAGTCGTATCCTGAATTCAACCTCTTTCTCTAAATGAATATAGCTATTGTTCCCTTTGTGCCCTCTTGTCCTACTGTCCCTTCTGTTGCCCATGCCAAAGACAGCTAGCTCCTTGAACAGCTTGGCCTGAATACAGATACTAGCGTGTCTGCAGCAGAGAAAAAAACAGCATTCCCCATCCAGAAATGCAAGGTCAAGAACAGAGAGCAAATTAGGTAGCTAAGGACTCAGGTCCTTAGTTGGTGTCCAGGGGCCACATTCTTTCCTTTCACCATCTCTGTAGGGACAGGAATACTTCCCTTCTGTCCTCAGAGGGTCAGGACTCAGAGAAACCACAGAGCAGCAGCTCAGGAAAGTGGTTCATGGAAATGCTGGCAAGAGAGAGGGGTTACAATGCCCTCCCTTGGGAGCAGGCTGCTCCCATCAGATCGTAACCTCTCTGGTATGTGGGCAGAGCTACCAGGTTAAGGTCCTCCCTAGGGTTTGCAAAACCCTCATGGGATCATGAGCCATACAGAACCGACCTGTGTGTCTCCAGAGTCTGTAATTAACACAGGCATTTTTGAGGAAATGCGTGGCCTCAGGCCCCACTCCCGGCTACCCCCATCCCACTATGCCTAGTATAGTCTAGCTGCCCTGGTACAATTCTCCCAGTATCTTGCAGGCCCCTATTTCCTATTCCTACTCTGCTCATCTGGCTCTCAGGAACCTTCTTGGCCTTCCCTTTCAGACCTCTACAGATGCCAGCCTTGTGGGAAAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTGCGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTTTGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAGCAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCCCTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAAGGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATGATCAGCTCAGCGGCCCAGCTGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTGATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGCCTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCTTCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATGATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCCAGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGACCAGTGGGTCAGCAGGCACGGCTGGCAGCCTTCTCTGCCCTGAGGGTCGAAGGTCGAGCAGGCCGGGGGTGTCCGGGAGGTCTTTGGGCATCGCGGTCTGGGGTTGGGACGTGTAAGCGCCTGGGAGAGCCTAGACCAGGCTCCGGGCTGCCAATAAAGAAGTGAAATGCGTATCTGGTCTCCTGTCGTGGGAGAGTGTGAGGTGTAACGGATTCAAGTCTGAACCCAGAGCCTGGAAAAGGCTGACCGCCCAGATTGACGTTGCTAGGCAACTCCGGAGGCGGGCCCAGCGCCAAAAGAACAGGGCGAGGCGTCGTCCCCGCATCCCATTGGCCGTTCTCTGCGGGGCCCCGCCCTCGGGGGCCGGAGCTAGAAGCTCTACGCTTCCGAGGCGCACCTCGTGGCCTGCACGCTTTGACGT

[0259] hT1R1 Predicted cds (SEQ ID NO 16)ATGCTGCTCTGCACGGCTCGCCTGGTCGGCCTGCAGCTTCTCATTTCCTGCTGCTGGGCCTT (SEQ IDNO 16) TGCCTGCCATAGCACGGAGTCTTCTCCTGACTTCACCCTCCCCGGAGATTACCTCGTGGCAGGCCTGTTCCCTCTCCATTCTGGCTGTCTGCAGGTGAGGCACAGACCGGAGGTGAGCGTGTGTGACAGGTCTTGTAGCTTCAATGAGCATGGCTACCACCTCUCCAGGGTATGCGGCTTGGGGTTGAGGAGATAAACAACTCCACGGCCCTGCTGCCCAACATCACCCTGGGGTACCAGCTGTATGATGTGTGTTCTGACTCTGCCAATGTGTATGCCACGCTGAGAGTGCTCTCCCTGCCAGGGCAACACCACATAGAGCTCCAAGGAGACCTTCTCCACTATTCCCCTACGGTGCTGGCAGTGATTGGGCCTGACAGCACCAACCGTGCTGCCACCACAGCCGCCCTGCTGAGCCCTTTCCTGGTGCCCATGATTAGCTATGCGGCCAGCAGCGAGACGCTCAGCGTGAAGCGGCAGTATCCCTCTTTCCTGCGCACCATCCCCAATGACAAGTACCAGGTGGAGACCATGGTGCTGCTGCTGCAGAAGTTCGGGTGGACCTGGATCTCTCTGGTTGGCAGCAGTGACGACTATGGGCAGCTAGGGGTGCAGGCACTGGAGAACCAGGCCACTGGTCAGGGGATCTGCATTGCTTTCAAGGACATCATGCCCTTCTCTGCCCAGGTGGGCGATGAGAGGATGCAGTGCCTCATGCGCCACCTGGCCCAGGCCGGGGCCACCGTCGTGGTTGTTTTTTCCAGCCGGCAGTTGGCCAGGGTGTTTTTCGAGTCCGTGGTGCTGACCAACCTGACTGGCAAGGTGTGGGTCGCCTCAGAAGCCTGGGCCCTCTCCAGGCACATCACTGGGGTGCCCGGGATCCAGCGCATTGGGATGGTGCTGGGCCTGGCCATCCAGAAGAGGGCTGTCCCTGGCCTGAAGGCGTTTGAAGAAGCCTATGCCCGGGCAGACAAGAAGGCCCCTAGGCCTTGCCACAAGGGCTCCTGGTGCAGCAGCAATCAGCTCTGCAGAGAATGCCAAGCTTTCATGGCACACACGATGCCCAAGCTCAAAGCCTTCTCCATGAGTTCTGCCTACAACGCATACCGGGCTGTGTATGCGGTGGCCCATGGCCTCCACCAGCTCCTGGGCTGTGCCTCTGGAGCTTGTTCCAGGGGCCGAGTCTACCCCTGGCAGCTTTTGGAGCAGATCCACAAGGTGCATTTCCTTCTACACAAGGACACTGTGGCGTTTAATGACAACAGAGATCCCCTCAGTAGCTATAACATAATTGCCTGGGACTGGAATGGACCCAAGTGGACCTTCACGGTCCTCGGTTCCTCCACATGGTCTCCAGTTCAGCTAAACATAAATGAGACCAAAATCCAGTGGCACGGAAGGACAACCAGGTGCCTAAGTCTGTGTGTTCCAGCGACTGTCTTGAAGGGCACCAGCGAGTGGTTACGGGTTTCCATCACTGCTGCTTTGAGTGTGTGCCCTGTGGGGCTGGGACCTTCCTCAACAAGAGTGACCTCTACAGATGCCAGCCTTGTGGGAAAGAAGAGTGGGCACCTGAGGGAAGCCAGACCTGCTTCCCGCGCACTGTGGTGTTTTTGGCTTTGCGTGAGCACACCTCTTGGGTGCTGCTGGCAGCTAACACGCTGCTGCTGCTGCTGCTGCTTGGGACTGCTGGCCTGTTTGCCTGGCACCTAGACACCCCTGTGGTGAGGTCAGCAGGGGGCCGCCTGTGCTTTCTTATGCTGGGCTCCCTGGCAGCAGGTAGTGGCAGCCTCTATGGCTTCTTTGGGGAACCCACAAGGCCTGCGTGCTTGCTACGCCAGGCCCTCTTTGCCCTTGGTTTCACCATCTTCCTGTCCTGCCTGACAGTTCGCTCATTCCAACTAATCATCATCTTCAAGTTTTCCACCAAGGTACCTACATTCTACCACGCCTGGGTCCAAAACCACGGTGCTGGCCTGTTTGTGATGATCAGCTCAGCGGCCCAGCTGCTTATCTGTCTAACTTGGCTGGTGGTGTGGACCCCACTGCCTGCTAGGGAATACCAGCGCTTCCCCCATCTGGTGATGCTTGAGTGCACAGAGACCAACTCCCTGGGCTTCATACTGGCCTTCCTCTACAATGGCCTCCTCTCCATCAGTGCCTTTGCCTGCAGCTACCTGGGTAAGGACTTGCCAGAGAACTACAACGAGGCCAAATGTGTCACCTTCAGCCTGCTCTTCAACTTCGTGTCCTGGATCGCCTTCTTCACCACGGCCAGCGTCTACGACGGCAAGTACCTGCCTGCGGCCAACATGATGGCTGGGCTGAGCAGCCTGAGCAGCGGCTTCGGTGGGTATTTTCTGCCTAAGTGCTACGTGATCCTCTGCCGCCCAGACCTCAACAGCACAGAGCACTTCCAGGCCTCCATTCAGGACTACACGAGGCGCTGCGGCTCCACCTGA

[0260] hT1R1 Conceptual Translation (SEQ ID NO 17)MLLCTARLVGLQLLISCCWAFACHSTESSPDFTLPGDYLLAGLFPLHSGCLQVRHRPEVTLCDR (SEQ IDNO 17) SCSFNEHGYHLFQAMRLGVEEINNSTALLPNITLGYQLYDVCSDSANVYATLRVLSLPGQHHIELQGDLLHYSPTVLAVIGPDSTNRAATTAALLSPFLVPMISYAASSETLSVKRQYPSFLRTIPNDKYQVETMVLLLQKFGWTWISLVGSSDDYGQLGVQALENQATGQGICIAFKDIMPFSAQVGDERMQCLMRHLAQAGATVVVVFSSRQLARVFFESVVLTNLTGKVWVASEAWALSRHITGVPGIQRIGMVLGVAIQKRAVPGLKAFEEAYARADKKAPRPCHKGSWCSSNQLCRECQAFMAHTMPKLKAFSMSSAYNAYRAVYAVAHGLHQLLGCASGACSRGRVYPWQLLEQIHKVHFLLHKDTVAFNDNRDPLSSYNIIAWDWNGPKWTFTVLGSSTWSPVQLNINETKIQWHGKDNQVPKSVCSSDCLEGHQRVVTGFHHCCFECVPCGAGTFLNKSDLYRCQPCGKEEWAPEGSQTCFPRTVVFLALREHTSWVLLAANTLLLLLLLGTAGLFAWHLDTPVVRSAGGRLCFLMLGSLAAGSGSLYGFFGEPTRPACLLRQALFALGFTIFLSCLTVRSFQLIIIFKFSTKVPTFYHAWVQNHGAGLFVMISSAAQLLICLTWLVVWTPLPAREYQRFPHLVMLECTETNSLGFILAFLYNGLLSISAFACSYLGKDLPENYNEAKCVTFSLLFNTVSWIAFFTTASVYDGKYLPAANMMAGLSSLSSGFGGYFLPKCYVILCRPDLNSTEHFQASIQDYTRRCGST

[0261] While the foregoing detailed description has described severalembodiments of the present invention, it is to be understood that theabove description is illustrative only and not limiting of the disclosedinvention. The invention is to be limited only by the claims whichfollow.

1 20 1 876 DNA Homo sapiens 1 agcctggcag tggcctcagg cagagtctgacgcgcacaaa ctttcaggcc caggaagcga 60 ggacaccact ggggccccag ggtgtggcaagtgaggatgg caagggtttt gctaaacaaa 120 tcctctgccc gctccccgcc ccgggctcactccatgtgag gccccagtcg gggcagccac 180 ctgccgtgcc tgttggaagt tgcctctgccatgctgggcc ctgctgtcct gggcctcagc 240 ctctgggctc tcctgcaccc tgggacgggggccccattgt gcctgtcaca gcaacttagg 300 atgaaggggg actacgtgct gggggggctgttccccctgg gcgaggccga ggaggctggc 360 ctccgcagcc ggacacggcc cagcagccctgtgtgcacca ggtacagagg tgggacggcc 420 tgggtcgggg tcagggtgac caggtctggggtgctcctga gctggggccg aggtggccat 480 ctgcggttct gtgtggcccc aggttctcctcaaacggcct gctctgggca ctggccatga 540 aaatggccgt ggaggagatc aacaacaagtcggatctgct gcccgggctg cgcctgggct 600 acgacctctt tgatacgtgc tcggagcctgtggtggccat gaagcccagc ctcatgttcc 660 tggccaaggc aggcagccgc gacatcgccgcctactgcaa ctacacgcag taccagcccc 720 gtgtgctggc tgtcatcggg ccccactcgtcagagctcgc catggtcacc ggcaagttct 780 tcagcttctt cctcatgccc cagtggggcgccccccacca tcacccaccc ccaaccaacc 840 cctgccccgt gggagcccct tgtgtcaggagaatgc 876 2 2687 DNA Homo sapiens 2 tacatgcacc ccacccagcc ctgccctgggagccctgtgt cagaagatgc tcttggcctt 60 gcaggtcagc tacggtgcta gcatggagctgctgagcgcc cgggagacct tcccctcctt 120 cttccgcacc gtgcccagcg accgtgtgcagctgacggcc gccgcggagc tgctgcagga 180 gttcggctgg aactgggtgg ccgccctgggcagcgacgac gagtacggcc ggcagggcct 240 gagcatcttc tcggccctgg ccgcggcacgcggcatctgc atcgcgcacg agggcctggt 300 gccgctgccc cgtgccgatg actcgcggctggggaaggtg caggacgtcc tgcaccaggt 360 gaaccagagc agcgtgcagg tggtgctgctgttcgcctcc gtgcacgccg cccacgccct 420 cttcaactac agcatcagca gcaggctctcgcccaaggtg tgggtggcca gcgaggcctg 480 gctgacctct gacctggtca tggggctgcccggcatggcc cagatgggca cggtgcttgg 540 cttcctccag aggggtgccc agctgcacgagttcccccag tacgtgaaga cgcacctggc 600 cctggccacc gacccggcct tctgctctgccctgggcgag agggagcagg gtctggagga 660 ggacgtggtg ggccagcgct gcccgcagtgtgactgcatc acgctgcaga acgtgagcgc 720 agggctaaat caccaccaga cgttctctgtctacgcagct gtgtatagcg tggcccaggc 780 cctgcacaac actcttcagt gcaacgcctcaggctgcccc gcgcaggacc ccgtgaagcc 840 ctggcaggtg agcccgggag atgggggtgtgctgtcctct gcatgtgccc aggccaccag 900 gcacggccac cacgcctgag ctggaggtggctggcggctc agccccgtcc cccgcccgca 960 gctcctggag aacatgtaca acctgaccttccacgtgggc gggctgccgc tgcggttcga 1020 cagcagcgga aacgtggaca tggagtacgacctgaagctg tgggtgtggc agggctcagt 1080 gcccaggctc cacgacgtgg gcaggttcaacggcagcctc aggacagagc gcctgaagat 1140 ccgctggcac acgtctgaca accaggtgaggtgagggtgg gtgtgccagg cgtgcccgtg 1200 gtagcccccg cggcagggcg cagcctgggggtgggggccg ttccagtctc ccgtgggcat 1260 gcccagccga gcagagccag accccaggcctgtgcgcaga agcccgtgtc ccggtgctcg 1320 cggcagtgcc aggagggcca ggtgcgccgggtcaaggggt tccactcctg ctgctacgac 1380 tgtgtggact gcgaggcggg cagctaccggcaaaacccag gtgagccgcc ttcccggcag 1440 gcgggggtgg gaacgcagca ggggagggtcctgccaagtc ctgactctga gaccagagcc 1500 cacagggtac aagacgaaca cccagcgcccttctcctctc tcacagacga catcgcctgc 1560 accttttgtg gccaggatga gtggtccccggagcgaagca cacgctgctt ccgccgcagg 1620 tctcggttcc tggcatgggg cgagccggctgtgctgctgc tgctcctgct gctgagcctg 1680 gcgctgggcc ttgtgctggc tgctttggggctgttcgttc accatcggga cagcccactg 1740 gttcaggcct cgggggggcc cctggcctgctttggcctgg tgtgcctggg cctggtctgc 1800 ctcagcgtcc tcctgttccc tggccagcccagccctgccc gatgcctggc ccagcagccc 1860 ttgtcccacc tcccgctcac gggctgcctgagcacactct tcctgcaggc ggccgagatc 1920 ttcgtggagt cagaactgcc tctgagctgggcagaccggc tgagtggctg cctgcggggg 1980 ccctgggcct ggctggtggt gctgctggccatgctggtgg aggtcgcact gtgcacctgg 2040 tacctggtgg ccttcccgcc ggaggtggtgacggactggc acatgctgcc cacggaggcg 2100 ctggtgcact gccgcacacg ctcctgggtcagcttcggcc tagcgcacgc caccaatgcc 2160 acgctggcct ttctctgctt cctgggcactttcctggtgc ggagccagcc gggctgctac 2220 aaccgtgccc gtggcctcac ctttgccatgctggcctact tcatcacctg ggtctccttt 2280 gtgcccctcc tggccaatgt gcaggtggtcctcaggcccg ccgtgcagat gggcgccctc 2340 ctgctctgtg tcctgggcat cctggctgccttccacctgc ccaggtgtta cctgctcatg 2400 cggcagccag ggctcaacac ccccgagttcttcctgggag ggggccctgg ggatgcccaa 2460 ggccagaatg acgggaacac aggaaatcaggggaaacatg agtgacccaa ccctgtgatc 2520 tcagccccgg tgaacccaga cttagctgcgatccccccca agccagcaat gacccgtgtc 2580 tcgctacaga gaccctcccg ctctaggttctgaccccagg ttgtctcctg accctgaccc 2640 cacagtgagc cctaggcctg gagcacgtggacacccctgt gaccatc 2687 3 2553 DNA Homo sapiens 3 atgctgggcc ctgctgtcctgggcctcagc ctctgggctc tcctgcaccc tgggacgggg 60 gccccattgt gcctgtcacagcaacttagg atgaaggggg actacgtgct gggggggctg 120 ttccccctgg gcgaggccgaggaggctggc ctccgcagcc ggacacggcc cagcagccct 180 gtgtgcacca ggttctcctcaaacggcctg ctctgggcac tggccatgaa aatggccgtg 240 gaggagatca acaacaagtcggatctgctg cccgggctgc gcctgggcta cgacctcttt 300 gatacgtgct cggagcctgtggtggccatg aagcccagcc tcatgttcct ggccaaggca 360 ggcagccgcg acatcgccgcctactgcaac tacacgcagt accagccccg tgtgctggct 420 gtcatcgggc cccactcgtcagagctcgcc atggtcaccg gcaagttctt cagcttcttc 480 ctcatgcccc actacggtgctagcatggag ctgctgagcg cccgggagac cttcccctcc 540 ttcttccgca ccgtgcccagcgaccgtgtg cagctgacgg ccgccgcgga gctgctgcag 600 gagttcggct ggaactgggtggccgccctg ggcagcgacg acgagtacgg ccggcagggc 660 ctgagcatct tctcggccctggccgcggca cgcggcatct gcatcgcgca cgagggcctg 720 gtgccgctgc cccgtgccgatgactcgcgg ctggggaagg tgcaggacgt cctgcaccag 780 gtgaaccaga gcagcgtgcaggtggtgctg ctgttcgcct ccgtgcacgc cgcccacgcc 840 ctcttcaact acagcatcagcagcaggctc tcgcccaagg tgtgggtggc cagcgaggcc 900 tggctgacct ctgacctggtcatggggctg cccggcatgg cccagatggg cacggtgctt 960 ggcttcctcc agaggggtgcccagctgcac gagttccccc agtacgtgaa gacgcacctg 1020 gccctggcca ccgacccggccttctgctct gccctgggcg agagggagca gggtctggag 1080 gaggacgtgg tgggccagcgctgcccgcag tgtgactgca tcacgctgca gaacgtgagc 1140 gcagggctaa atcaccaccagacgttctct gtctacgcag ctgtgtatag cgtggcccag 1200 gccctgcaca acactcttcagtgcaacgcc tcaggctgcc ccgcgcagga ccccgtgaag 1260 ccctggcagc tcctggagaacatgtacaac ctgaccttcc acgtgggcgg gctgccgctg 1320 cggttcgaca gcagcggaaacgtggacatg gagtacgacc tgaagctgtg ggtgtggcag 1380 ggctcagtgc ccaggctccacgacgtgggc aggttcaacg gcagcctcag gacagagcgc 1440 ctgaagatcc gctggcacacgtctgacaac cagaagcccg tgtcccggtg ctcgcggcag 1500 tgccaggagg gccaggtgcgccgggtcaag gggttccact cctgctgcta cgactgtgtg 1560 gactgcgagg cgggcagctaccggcaaaac ccagacgaca tcgcctgcac cttttgtggc 1620 caggatgagt ggtccccggagcgaagcaca cgctgcttcc gccgcaggtc tcggttcctg 1680 gcatggggcg agccggctgtgctgctgctg ctcctgctgc tgagcctggc gctgggcctt 1740 gtgctggctg ctttggggctgttcgttcac catcgggaca gcccactggt tcaggcctcg 1800 ggggggcccc tggcctgctttggcctggtg tgcctgggcc tggtctgcct cagcgtcctc 1860 ctgttccctg gccagcccagccctgcccga tgcctggccc agcagccctt gtcccacctc 1920 ccgctcacgg gctgcctgagcacactcttc ctgcaggcgg ccgagatctt cgtggagtca 1980 gaactgcctc tgagctgggcagaccggctg agtggctgcc tgcgggggcc ctgggcctgg 2040 ctggtggtgc tgctggccatgctggtggag gtcgcactgt gcacctggta cctggtggcc 2100 ttcccgccgg aggtggtgacggactggcac atgctgccca cggaggcgct ggtgcactgc 2160 cgcacacgct cctgggtcagcttcggccta gcgcacgcca ccaatgccac gctggccttt 2220 ctctgcttcc tgggcactttcctggtgcgg agccagccgg gctgctacaa ccgtgcccgt 2280 ggcctcacct ttgccatgctggcctacttc atcacctggg tctcctttgt gcccctcctg 2340 gccaatgtgc aggtggtcctcaggcccgcc gtgcagatgg gcgccctcct gctctgtgtc 2400 ctgggcatcc tggctgccttccacctgccc aggtgttacc tgctcatgcg gcagccaggg 2460 ctcaacaccc ccgagttcttcctgggaggg ggccctgggg atgcccaagg ccagaatgac 2520 gggaacacag gaaatcaggggaaacatgag tga 2553 4 850 PRT Homo sapiens 4 Met Leu Gly Pro Ala Val LeuGly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly Thr Gly Ala ProLeu Cys Leu Ser Gln Gln Leu Arg Met Lys 20 25 30 Gly Asp Tyr Val Leu GlyGly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly Leu Arg Ser ArgThr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser Ser Asn Gly LeuLeu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu Ile Asn AsnLys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe AspThr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 Ser Leu Met PheLeu Ala Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr 115 120 125 Cys Asn TyrThr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro 130 135 140 His SerSer Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160Leu Met Pro His Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala Arg Glu 165 170175 Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val Gln Leu 180185 190 Thr Ala Ala Ala Glu Leu Leu Gln Glu Phe Gly Trp Asn Trp Val Ala195 200 205 Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Ser IlePhe 210 215 220 Ser Ala Leu Ala Ala Ala Arg Gly Ile Cys Ile Ala His GluGly Leu 225 230 235 240 Val Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu GlyLys Val Gln Asp 245 250 255 Val Leu His Gln Val Asn Gln Ser Ser Val GlnVal Val Leu Leu Phe 260 265 270 Ala Ser Val His Ala Ala His Ala Leu PheAsn Tyr Ser Ile Ser Ser 275 280 285 Arg Leu Ser Pro Lys Val Trp Val AlaSer Glu Ala Trp Leu Thr Ser 290 295 300 Asp Leu Val Met Gly Leu Pro GlyMet Ala Gln Met Gly Thr Val Leu 305 310 315 320 Gly Phe Leu Gln Arg GlyAla Gln Leu His Glu Phe Pro Gln Tyr Val 325 330 335 Lys Thr His Leu AlaLeu Ala Thr Asp Pro Ala Phe Cys Ser Ala Leu 340 345 350 Gly Glu Arg GluGln Gly Leu Glu Glu Asp Val Val Gly Gln Arg Cys 355 360 365 Pro Gln CysAsp Cys Ile Thr Leu Gln Asn Val Ser Ala Gly Leu Asn 370 375 380 His HisGln Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val Ala Gln 385 390 395 400Ala Leu His Asn Thr Leu Gln Cys Asn Ala Ser Gly Cys Pro Ala Gln 405 410415 Asp Pro Val Lys Pro Trp Gln Leu Leu Glu Asn Met Tyr Asn Leu Thr 420425 430 Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly Asn Val435 440 445 Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gln Gly Ser ValPro 450 455 460 Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg ThrGlu Arg 465 470 475 480 Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln LysPro Val Ser Arg 485 490 495 Cys Ser Arg Gln Cys Gln Glu Gly Gln Val ArgArg Val Lys Gly Phe 500 505 510 His Ser Cys Cys Tyr Asp Cys Val Asp CysGlu Ala Gly Ser Tyr Arg 515 520 525 Gln Asn Pro Asp Asp Ile Ala Cys ThrPhe Cys Gly Gln Asp Glu Trp 530 535 540 Ser Pro Glu Arg Ser Thr Arg CysPhe Arg Arg Arg Ser Arg Phe Leu 545 550 555 560 Ala Trp Gly Glu Pro AlaVal Leu Leu Leu Leu Leu Leu Leu Ser Leu 565 570 575 Ala Leu Gly Leu ValLeu Ala Ala Leu Gly Leu Phe Val His His Arg 580 585 590 Asp Ser Pro LeuVal Gln Ala Ser Gly Gly Pro Leu Ala Cys Phe Gly 595 600 605 Leu Val CysLeu Gly Leu Val Cys Leu Ser Val Leu Leu Phe Pro Gly 610 615 620 Gln ProSer Pro Ala Arg Cys Leu Ala Gln Gln Pro Leu Ser His Leu 625 630 635 640Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gln Ala Ala Glu Ile 645 650655 Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asp Arg Leu Ser Gly 660665 670 Cys Leu Arg Gly Pro Trp Ala Trp Leu Val Val Leu Leu Ala Met Leu675 680 685 Val Glu Val Ala Leu Cys Thr Trp Tyr Leu Val Ala Phe Pro ProGlu 690 695 700 Val Val Thr Asp Trp His Met Leu Pro Thr Glu Ala Leu ValHis Cys 705 710 715 720 Arg Thr Arg Ser Trp Val Ser Phe Gly Leu Ala HisAla Thr Asn Ala 725 730 735 Thr Leu Ala Phe Leu Cys Phe Leu Gly Thr PheLeu Val Arg Ser Gln 740 745 750 Pro Gly Cys Tyr Asn Arg Ala Arg Gly LeuThr Phe Ala Met Leu Ala 755 760 765 Tyr Phe Ile Thr Trp Val Ser Phe ValPro Leu Leu Ala Asn Val Gln 770 775 780 Val Val Leu Arg Pro Ala Val GlnMet Gly Ala Leu Leu Leu Cys Val 785 790 795 800 Leu Gly Ile Leu Ala AlaPhe His Leu Pro Arg Cys Tyr Leu Leu Met 805 810 815 Arg Gln Pro Gly LeuAsn Thr Pro Glu Phe Phe Leu Gly Gly Gly Pro 820 825 830 Gly Asp Ala GlnGly Gln Asn Asp Gly Asn Thr Gly Asn Gln Gly Lys 835 840 845 His Glu 8505 23 DNA Artificial Sequence modified_base (3) a, c, t, g, other orunknown 5 cgnttyytng cntggggnga rcc 23 6 23 DNA Artificial Sequencemodified_base (3) a, c, t, g, other or unknown 6 cgngcncgrt trtarcanccngg 23 7 9 PRT Homo sapiens 7 Arg Phe Leu Ala Trp Gly Glu Pro Ala 1 5 88 PRT Homo sapiens 8 Pro Gly Cys Tyr Asn Arg Ala Arg 1 5 9 552 DNA Mussp. 9 gtgctgtcac tcctcctgct gctttgcctg gtgctgggtc tagcactggc tgctctgggg60 ctctctgtcc accactggga cagccctctt gtccaggcct caggcggctc acagttctgc 120tttggcctga tctgcctagg cctcttctgc ctcagtgtcc ttctgttccc aggacggcca 180agctctgcca gctgccttgc acaacaacca atggctcacc tccctctcac aggctgcctg 240agcacactct tcctgcaagc agctgagacc tttgtggagt ctgagctgcc actgagctgg 300gcaaactggc tatgcagcta ccttcgggac tctggcctgc tagtggtact gttggccact 360tttgtggagg cagcactatg tgcctggtat ttgaccgctt caccagaagt ggtgacagac 420tggtcagtgc tgcccacaga ggtactggag cactgccacg tgcgttcctg ggtcaacctg 480ggcttggtgc acatcaccaa tgcaatggta gcttttctct gctttctggg cactttcctg 540gtacaagacc ag 552 10 184 PRT Mus sp. 10 Val Leu Ser Leu Leu Leu Leu LeuCys Leu Val Leu Gly Leu Ala Leu 1 5 10 15 Ala Ala Leu Gly Leu Ser ValHis His Trp Asp Ser Pro Leu Val Gln 20 25 30 Ala Ser Gly Gly Ser Gln PheCys Phe Gly Leu Ile Cys Leu Gly Leu 35 40 45 Phe Cys Leu Ser Val Leu LeuPhe Pro Gly Arg Pro Ser Ser Ala Ser 50 55 60 Cys Leu Ala Gln Gln Pro MetAla His Leu Pro Leu Thr Gly Cys Leu 65 70 75 80 Ser Thr Leu Phe Leu GlnAla Ala Glu Thr Phe Val Glu Ser Glu Leu 85 90 95 Pro Leu Ser Trp Ala AsnTrp Leu Cys Ser Tyr Leu Arg Asp Ser Gly 100 105 110 Leu Leu Val Val LeuLeu Ala Thr Phe Val Glu Ala Ala Leu Cys Ala 115 120 125 Trp Tyr Leu ThrAla Ser Pro Glu Val Val Thr Asp Trp Ser Val Leu 130 135 140 Pro Thr GluVal Leu Glu His Cys His Val Arg Ser Trp Val Asn Leu 145 150 155 160 GlyLeu Val His Ile Thr Asn Ala Met Val Ala Phe Leu Cys Phe Leu 165 170 175Gly Thr Phe Leu Val Gln Asp Gln 180 11 558 DNA Rattus sp. 11 gtgctgtcacttctcctgct gctttgcctg gtgctgggcc tgacactggc tgccctgggg 60 ctctttgtccactactggga cagccctctt gttcaggcct caggtgggtc actgttctgc 120 tttggcctgatctgcctagg cctcttctgc ctcagtgtcc ttctgttccc aggacgacca 180 cgctctgccagctgccttgc ccaacaacca atggctcacc tccctctcac aggctgcctg 240 agcacactcttcctgcaagc agccgagatc tttgtggagt ctgagctgcc actgagttgg 300 gcaaactggctctgcagcta ccttcggggc ccctgggctt ggctggtggt actgctggcc 360 actcttgtggaggctgcact atgtgcctgg tacttgatgg ctttccctcc agaggtggtg 420 acagattggcaggtgctgcc cacggaggta ctggaacact gccgcatgcg ttcctgggtc 480 agcctgggcttggtgcacat caccaatgca ggggtagctt tcctctgctt tctgggcact 540 ttcctggtacaaagccag 558 12 186 PRT Rattus sp. 12 Val Leu Ser Leu Leu Leu Leu LeuCys Leu Val Leu Gly Leu Thr Leu 1 5 10 15 Ala Ala Leu Gly Leu Phe ValHis Tyr Trp Asp Ser Pro Leu Val Gln 20 25 30 Ala Ser Gly Gly Ser Leu PheCys Phe Gly Leu Ile Cys Leu Gly Leu 35 40 45 Phe Cys Leu Ser Val Leu LeuPhe Pro Gly Arg Pro Arg Ser Ala Ser 50 55 60 Cys Leu Ala Gln Gln Pro MetAla His Leu Pro Leu Thr Gly Cys Leu 65 70 75 80 Ser Thr Leu Phe Leu GlnAla Ala Glu Ile Phe Val Glu Ser Glu Leu 85 90 95 Pro Leu Ser Trp Ala AsnTrp Leu Cys Ser Tyr Leu Arg Gly Pro Trp 100 105 110 Ala Trp Leu Val ValLeu Leu Ala Thr Leu Val Glu Ala Ala Leu Cys 115 120 125 Ala Trp Tyr LeuMet Ala Phe Pro Pro Glu Val Val Thr Asp Trp Gln 130 135 140 Val Leu ProThr Glu Val Leu Glu His Cys Arg Met Arg Ser Trp Val 145 150 155 160 SerLeu Gly Leu Val His Ile Thr Asn Ala Gly Val Ala Phe Leu Cys 165 170 175Phe Leu Gly Thr Phe Leu Val Gln Ser Gln 180 185 13 2577 DNA Rattus sp.13 atgccgggtt tggctatctt gggcctcagt ctggctgctt tcctggagct tgggatgggg 60tcctctttgt gtctgtcaca gcaattcaag gcacaagggg actatatatt gggtggacta 120tttcccctgg gcacaactga ggaggccact ctcaaccaga gaacacagcc caacggcatc 180ctatgtacca ggttctcgcc ccttggtttg ttcctggcca tggctatgaa gatggctgta 240gaggagatca acaatggatc tgccttgctc cctgggctgc gactgggcta tgacctgttt 300gacacatgct cagagccagt ggtcaccatg aagcccagcc tcatgttcat ggccaaggtg 360ggaagtcaaa gcattgctgc ctactgcaac tacacacagt accaaccccg tgtgctggct 420gtcattggtc cccactcatc agagcttgcc ctcattacag gcaagttctt cagcttcttc 480ctcatgccac aggtcagcta tagtgccagc atggatcggc taagtgaccg ggaaacattt 540ccatccttct tccgcacagt gcccagtgac cgggtgcagc tgcaggccgt tgtgacactg 600ttgcagaatt tcagctggaa ctgggtggct gccttaggta gtgatgatga ctatggccgg 660gaaggtctga gcatcttttc tggtctggcc aactcacgag gtatctgcat tgcacacgag 720ggcctggtgc cacaacatga cactagtggc caacaattgg gcaaggtggt ggatgtgcta 780cgccaagtga accaaagcaa agtacaggtg gtggtgctgt ttgcatctgc ccgtgctgtc 840tactcccttt ttagctacag catccttcat gacctctcac ccaaggtatg ggtggccagt 900gagtcctggc tgacctctga cctggtcatg acacttccca atattgcccg tgtgggcact 960gttcttgggt ttctgcagcg cggtgcccta ctgcctgaat tttcccatta tgtggagact 1020cgccttgccc tagctgctga cccaacattc tgtgcctccc tgaaagctga gttggatctg 1080gaggagcgcg tgatggggcc acgctgttca caatgtgact acatcatgct acagaacctg 1140tcatctgggc tgatgcagaa cctatcagct gggcagttgc accaccaaat atttgcaacc 1200tatgcagctg tgtacagtgt ggctcaggcc cttcacaaca ccctgcagtg caatgtctca 1260cattgccaca catcagagcc tgttcaaccc tggcagctcc tggagaacat gtacaatatg 1320agtttccgtg ctcgagactt gacactgcag tttgatgcca aagggagtgt agacatggaa 1380tatgacctga agatgtgggt gtggcagagc cctacacctg tactacatac tgtaggcacc 1440ttcaacggca cccttcagct gcagcactcg aaaatgtatt ggccaggcaa ccaggtgcca 1500gtctcccagt gctcccggca gtgcaaagat ggccaggtgc gcagagtaaa gggctttcat 1560tcctgctgct atgactgtgt ggactgcaag gcagggagct accggaagca tccagatgac 1620ttcacctgta ctccatgtgg caaggatcag tggtccccag aaaaaagcac aacctgctta 1680cctcgcaggc ccaagtttct ggcttggggg gagccagctg tgctgtcact tctcctgctg 1740ctttgcctgg tgctgggcct gacactggct gccctggggc tctttgtcca ctactgggac 1800agccctcttg ttcaggcctc aggtgggtca ctgttctgct ttggcctgat ctgcctaggc 1860ctcttctgcc tcagtgtcct tctgttccca ggacgaccac gctctgccag ctgccttgcc 1920caacaaccaa tggctcacct ccctctcaca ggctgcctga gcacactctt cctgcaagca 1980gccgagatct ttgtggagtc tgagctgcca ctgagttggg caaactggct ctgcagctac 2040cttcggggcc cctgggcttg gctggtggta ctgctggcca ctcttgtgga ggctgcacta 2100tgtgcctggt acttgatggc tttccctcca gaggtggtga cagattggca ggtgctgccc 2160acggaggtac tggaacactg ccgcatgcgt tcctgggtca gcctgggctt ggtgcacatc 2220accaatgcag tgttagcttt cctctgcttt ctgggcactt tcctggtaca gagccagcct 2280ggtcgctata accgtgcccg tggcctcacc ttcgccatgc tagcttattt catcatctgg 2340gtctcttttg tgcccctcct ggctaatgtg caggtggcct accagccagc tgtgcagatg 2400ggtgctatct tattctgtgc cctgggcatc ctggccacct tccacctgcc caaatgctat 2460gtacttctgt ggctgccaga gctcaacacc caggagttct tcctgggaag gagccccaag 2520gaagcatcag atgggaatag tggtagtagt gaggcaactc ggggacacag tgaatga 2577 14858 PRT Rattus sp. 14 Met Pro Gly Leu Ala Ile Leu Gly Leu Ser Leu AlaAla Phe Leu Glu 1 5 10 15 Leu Gly Met Gly Ser Ser Leu Cys Leu Ser GlnGln Phe Lys Ala Gln 20 25 30 Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro LeuGly Thr Thr Glu Glu 35 40 45 Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn GlyIle Leu Cys Thr Arg 50 55 60 Phe Ser Pro Leu Gly Leu Phe Leu Ala Met AlaMet Lys Met Ala Val 65 70 75 80 Glu Glu Ile Asn Asn Gly Ser Ala Leu LeuPro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu ProVal Val Thr Met Lys Pro 100 105 110 Ser Leu Met Phe Met Ala Lys Val GlySer Gln Ser Ile Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gln Tyr Gln ProArg Val Leu Ala Val Ile Gly Pro 130 135 140 His Ser Ser Glu Leu Ala LeuIle Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gln ValSer Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp 165 170 175 Arg Glu Thr PhePro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gln Leu GlnAla Val Val Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp 195 200 205 Val AlaAla Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210 215 220 IlePhe Ser Gly Leu Ala Asn Ser Arg Gly Ile Cys Ile Ala His Glu 225 230 235240 Gly Leu Val Pro Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val 245250 255 Val Asp Val Leu Arg Gln Val Asn Gln Ser Lys Val Gln Val Val Val260 265 270 Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr SerIle 275 280 285 Leu His Asp Leu Ser Pro Lys Val Trp Val Ala Ser Glu SerTrp Leu 290 295 300 Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile Ala ArgVal Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gln Arg Gly Ala Leu LeuPro Glu Phe Ser His 325 330 335 Tyr Val Glu Thr Arg Leu Ala Leu Ala AlaAsp Pro Thr Phe Cys Ala 340 345 350 Ser Leu Lys Ala Glu Leu Asp Leu GluGlu Arg Val Met Gly Pro Arg 355 360 365 Cys Ser Gln Cys Asp Tyr Ile MetLeu Gln Asn Leu Ser Ser Gly Leu 370 375 380 Met Gln Asn Leu Ser Ala GlyGln Leu His His Gln Ile Phe Ala Thr 385 390 395 400 Tyr Ala Ala Val TyrSer Val Ala Gln Ala Leu His Asn Thr Leu Gln 405 410 415 Cys Asn Val SerHis Cys His Thr Ser Glu Pro Val Gln Pro Trp Gln 420 425 430 Leu Leu GluAsn Met Tyr Asn Met Ser Phe Arg Ala Arg Asp Leu Thr 435 440 445 Leu GlnPhe Asp Ala Lys Gly Ser Val Asp Met Glu Tyr Asp Leu Lys 450 455 460 MetTrp Val Trp Gln Ser Pro Thr Pro Val Leu His Thr Val Gly Thr 465 470 475480 Phe Asn Gly Thr Leu Gln Leu Gln His Ser Lys Met Tyr Trp Pro Gly 485490 495 Asn Gln Val Pro Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln500 505 510 Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys ValAsp 515 520 525 Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe ThrCys Thr 530 535 540 Pro Cys Gly Lys Asp Gln Trp Ser Pro Glu Lys Ser ThrThr Cys Leu 545 550 555 560 Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly GluPro Ala Val Leu Ser 565 570 575 Leu Leu Leu Leu Leu Cys Leu Val Leu GlyLeu Thr Leu Ala Ala Leu 580 585 590 Gly Leu Phe Val His Tyr Trp Asp SerPro Leu Val Gln Ala Ser Gly 595 600 605 Gly Ser Leu Phe Cys Phe Gly LeuIle Cys Leu Gly Leu Phe Cys Leu 610 615 620 Ser Val Leu Leu Phe Pro GlyArg Pro Arg Ser Ala Ser Cys Leu Ala 625 630 635 640 Gln Gln Pro Met AlaHis Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu 645 650 655 Phe Leu Gln AlaAla Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser 660 665 670 Trp Ala AsnTrp Leu Cys Ser Tyr Leu Arg Gly Pro Trp Ala Trp Leu 675 680 685 Val ValLeu Leu Ala Thr Leu Val Glu Ala Ala Leu Cys Ala Trp Tyr 690 695 700 LeuMet Ala Phe Pro Pro Glu Val Val Thr Asp Trp Gln Val Leu Pro 705 710 715720 Thr Glu Val Leu Glu His Cys Arg Met Arg Ser Trp Val Ser Leu Gly 725730 735 Leu Val His Ile Thr Asn Ala Val Leu Ala Phe Leu Cys Phe Leu Gly740 745 750 Thr Phe Leu Val Gln Ser Gln Pro Gly Arg Tyr Asn Arg Ala ArgGly 755 760 765 Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Ile Trp Val SerPhe Val 770 775 780 Pro Leu Leu Ala Asn Val Gln Val Ala Tyr Gln Pro AlaVal Gln Met 785 790 795 800 Gly Ala Ile Leu Phe Cys Ala Leu Gly Ile LeuAla Thr Phe His Leu 805 810 815 Pro Lys Cys Tyr Val Leu Leu Trp Leu ProGlu Leu Asn Thr Gln Glu 820 825 830 Phe Phe Leu Gly Arg Ser Pro Lys GluAla Ser Asp Gly Asn Ser Gly 835 840 845 Ser Ser Glu Ala Thr Arg Gly HisSer Glu 850 855 15 8194 DNA Homo sapiens modified_base (1251)..(1300) a,c, t, g, other or unknown 15 gagaatctcg cgagatcccg tcggtccgcc ccgctgccctcccagctgcc gaaaagaggg 60 gcctccgagc cgccggcgcc ctctgccggc aacctccggaagcacactag gaggttccag 120 ccgatctggt cgaggggctc cacggaggac tccatttacgttacgcaaat tccctacccc 180 agccggccgg agagagaaag ccagaaacct cgcgaccagccatgggccac ctctccggaa 240 aaacaccggg atattttttt tctcctgcag aaaaagctttaggattggca gtttaaacaa 300 aacatgtcta tttgcatacc ttcggtttgc atgcatttgtttcgaagtga gcaaccctgg 360 gtaacaaggc gaaagtatat gacaatttgc tcagaatcttaatgtcagaa aactggagac 420 tggggcaggg gggtgtcgac tcaaagctgt gtctcatttagtaaactgag gcccaggtaa 480 aaagttctga aacctcgcaa cacccggaga aattgtgttccagcctccca cctcgcccca 540 aaatgccaga gctccttttc taagccaggt gaagtcacagagcgtggaca gaacccacaa 600 ccgtccagag gaagggtcac tgggtgccac ctggtttgcatctgtgcctt cgtcctgccc 660 agttcctgag tgggaccgca ggcccggaat gtcaaggcaaacagtcctgc ttcagccact 720 gggctccagt cccacccctt ttgggggcct gaagttaggaagcatccggc agctgccttc 780 tatttaagca actggcctcc ttagaggcca ctccttggccatgccaggcg cgggcatctg 840 gccagcatgc tgctctgcac ggctcgcctg gtcggcctgcagcttctcat ttcctgctgc 900 tgggcctttg cctgccatag cacggagtct tctcctgacttcaccctccc cggagattac 960 ctcctggcag gcctgttccc tctccattct ggctgtctgcaggtgaggca cagacccgag 1020 gtgaccctgt gtgacaggtg agtgaggggc cagcagagccacacttagtg ggacccctgg 1080 ctatagggcc cctctggctg ccatcctcca aacaggaccttgcctctgcc tttgcccctt 1140 gaactgtccc caggccttgt tcatcaatcc acttgccacctaagtgctgg ctagaccttc 1200 ctagacactt cggccagttt ccaattattt cacccttgctgttagaatgt nnnnnnnnnn 1260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnaattccttaa actaaatttc 1320 tcactttctc tctctctctg gaaaacactg actaatgtagcaggtttctc tgctccagga 1380 cttcaggacc ttttcgatgc taataagttt ctccatcagggccagcttgt tcctcctact 1440 gagcttgaga gcccttgttg aagttgtggt ttgggggactggaccgatga cctcaaaggt 1500 tccctttgct cccaagcctc agagtctagg aggccagagggtctcagcag gcctttgtcc 1560 ttctcagctg tctcttactg gctttctcca caggtcttgtagcttcaatg agcatggcta 1620 ccacctcttc caggctatgc ggcttggggt tgaggagataaacaactcca cggccctgct 1680 gcccaacatc accctggggt accagctgta tgatgtgtgttctgactctg ccaatgtgta 1740 tgccacgctg agagtgctct ccctgccagg gcaacaccacatagagctcc aaggagacct 1800 tctccactat tcccctacgg tgctggcagt gattgggcctgacagcacca accgtgctgc 1860 caccacagcc gccctgctga gccctttcct ggtgcccatggtaagctgga gcctcagacc 1920 tttgcccatc tcccttcagg caagtctggg nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnn gccaccatgc ccggctaatttttttgtatt tttagtagag 2040 acggggtttc accgtgttag ccaggctggt cgcaaactcctaacctcgtg atccacccac 2100 ctcggcctcc caatgtgctg ggattacagg tgtgagccactgcacccggc cataatgtat 2160 taatataata aaataattat acaactcacc ataatgtagaatcagtggga gccctgagct 2220 tgttttccta caactagatg gtcccatctg ggggtgatgggagacagtga cagatcatca 2280 gacattagat tctcataagt agcgtgcaac ccagatccctcgcatgtgca gttcacagta 2340 gggttcaagc tcctacaaga atctgatgct gctgctgatctgacaggagg ggagcagctg 2400 taaatacaga tgaagcttcg cttactcacc agctgctcacctcctcctgt gaggcccggt 2460 tcctaacagg ccactgacct aacttctgcc ctgacctacacatgcttctc ttcttccttg 2520 caaactgcct ccagtggaag tccctgaagg tccccaaacacacgggacta tttcactcct 2580 atgcaggttt tgtctccttt gcttggaatg catcccctcaccccttgtcc ccaggcagat 2640 tcccacccct cccccagaac ctgccccagt ggagccttcgcaggtgattt gtcagtttca 2700 caggctgagg ggtgctctcc tggtctcccc ggctccctgtatccccacac ccagcacagg 2760 gccaggcact gggggggcct tcagtggaga ctgaaatggctgaacgggac ctcccataga 2820 ttagctatgc ggccagcagc gagacgctca gcgtgaagcggcagtatccc tctttcctgc 2880 gcaccatccc caatgacaag taccaggtgg agaccatggtgctgctgctg cagaagttcg 2940 ggtggacctg gatctctctg gttggcagca gtgacgactatgggcagcta ggggtgcagg 3000 cactggagaa ccaggccact ggtcagggga tctgcattgctttcaaggac atcatgccct 3060 tctctgccca ggtgggcgat gagaggatgc agtgcctcatgcgccacctg gcccaggccg 3120 gggccaccgt cgtggttgtt ttttccagcc ggcagttggccagggtgttt ttcgagtccg 3180 tggtgctgac caacctgact ggcaaggtgt gggtcgcctcagaagcctgg gccctctcca 3240 ggcacatcac tggggtgccc gggatccagc gcattgggatggtgctgggc gtggccatcc 3300 agaagagggc tgtccctggc ctgaaggcgt ttgaagaagcctatgcccgg gcagacaaga 3360 aggcccctag gccttgccac aagggctcct ggtgcagcagcaatcagctc tgcagagaat 3420 gccaagcttt catggcacac acgatgccca agctcaaagccttctccatg agttctgcct 3480 acaacgcata ccgggctgtg tatgcggtgg cccatggcctccaccagctc ctgggctgtg 3540 cctctggagc ttgttccagg ggccgagtct acccctggcaggtaagagag cccaccccag 3600 cacctcctgt cagggagaac agccaatcct gagatgagcagagtgggcac tctccggtca 3660 ctctaaatgc caagggggat aaatgccact aacttgaggttttttgtttt gttttgtttt 3720 gttttttgag acagtctggc tctgtcaccc aggctgcagtgtagtgatgc gatctcggct 3780 ctctgcaact tccacctcct gggttcaagt gattctcttgcctcggcctc ctgagtagct 3840 gggattacag gcacccacca ccatgcctgg ataatttttcttttcttttt tttttttttg 3900 agatagagtc tcgctctgtt gcccaggctg gaatgcagtggtgcgatctt ggctcactgt 3960 gagctccgcc tcccaggttc actccattcc cctgcctcagcctcccaagt aggtgggact 4020 acgggcgccc gccaccacgc ccagctaatt ttttttgtattttgagtaga gacggggttt 4080 caccatgtta gccaggatgg tctcaatctc ctgaccttgtcatccgccca cctcgtcctc 4140 ccaaagtgct gggattacag gcgtgagcca ccgcacccggcctaattttt gtatttttag 4200 tagagatggg gtttcaccat gttggccagg ctggtctcgaactcctggca tcaagtgatc 4260 ctcctgcttc ggcctcccaa agtgctggga ttacaggcattagctctctt ctcttagaca 4320 gatctttctc tctgatcctt gccttctctc acccactgtgtcttggaagt gtcaagtgat 4380 aagatccagg gctaaaactg tctgtaaagg agtgtttgttagaggcctcc tctcaggagg 4440 ttggtgggga agattgaggg gcttcctaag aaggaagggacgagaccttc ctgatgggct 4500 gaaaccacca ggacggaaac ccaggaaggc cccaggcccttgcttctggg accatgtggg 4560 tctgtgctgt ctgtggtggc ttcatgatac gcgtttctttcagcttttgg agcagatcca 4620 caaggtgcat ttccttctac acaaggacac tgtggcgtttaatgacaaca gagatcccct 4680 cagtagctat aacataattg cctgggactg gaatggacccaagtggacct tcacggtcct 4740 cggttcctcc acatggtctc cagttcagct aaacataaatgagaccaaaa tccagtggca 4800 cggaaaggac aaccaggtaa tggggatgtg gctactcaccatgtaactgg cttatgggca 4860 acctagagcc tgggggtgat gctgacacag tgtacagggagcaggagggg ggccccaggg 4920 gtccagctgc caccactcta cccatcctgg ccagggaagcagggaagaca ctccgtaggc 4980 gagtgtgcag atgccctggg gcggaagttc acacgaccaggggccctgcc ctgggagtga 5040 gccctgaggg cagatgcaca gagattctgt tttctgttccacatgtgagc tgtcctttga 5100 cttgggcccc tacgtgtggc ccctctggct tcttacaggtgcctaagtct gtgtgttcca 5160 gcgactgtct tgaagggcac cagcgagtgg ttacgggtttccatcactgc tgctttgagt 5220 gtgtgccctg tggggctggg accttcctca acaagagtggtgagtgggca atggagcagg 5280 cgagctaccc agcactcccg ggggctgcac ggtggagggagggcctccct tgggccccat 5340 gtgccctgcc ccagaaccaa ggcccagtca ctgggctgccagttagcttc aggttggagg 5400 acacctgcta ccagacagaa ttctgatcaa gagaatcagccactgggtgc ggtggctcat 5460 gcctgtaatc ccagcacttt gggaggctga ggcgggtggatcacttgagg tcgggagttc 5520 gagaccagcc tggccaacat ggtgaaaccc catctctaccaaaaatataa aaaattagct 5580 gggtgtggtg gcgcgtgcct gtaatcccag ctactcgggaggctgaggca ggagaatcac 5640 ttgaacccag gaggcggagg ttgcagtgag ccaagatgcattccagcctg gaccacaaag 5700 cgagaattcg tccccccaaa aaaagaaagg aggccgggcgcggtggctca cacctgtaat 5760 cccagcactt tgggaggccg aggtgggtgg atcacctgaggtcaggagtt cgagaccagc 5820 ctgaccaaca tggtgaaacc ccatctctac taaaaatacaaaaaaagtta gccgggcgtt 5880 gtggcgtgtg cctgtaattc cagctactcg ggaggctgaggcaggagaat tgcttgaacc 5940 cgggaggcgg aggttgcagt gagccaagat tgcaccattgcactccagcc tgggcgacaa 6000 gagaaaaact ctgtctcaaa aaaaaagaaa gaaagaaagaattagccaac tgaaagcctt 6060 agactgaggt gtgtcctctg ttagagagct gtcatcacaactcctacaaa agcagtcgta 6120 tcctgaattc aacctctttc tctaaatgaa tatagctattgttccctttg tgccctcttg 6180 tcctactgtc ccttctgttg cccatgccaa agacagctagctccttgaac agcttggcct 6240 gaatacagat actagcgtgt ctgcagcaga gaaaaaaacagcattcccca tccagaaatg 6300 caaggtcaag aacagagagc aaattaggta gctaaggactcaggtcctta gttggtgtcc 6360 aggggccaca ttctttcctt tcaccatctc tgtagggacaggaatacttc ccttctgtcc 6420 tcagagggtc aggactcaga gaaaccacag agcagcagctcaggaaagtg gttcatggaa 6480 atgctggcaa gagagagggg ttacaatgcc ctcccttgggagcaggctgc tcccatcaga 6540 tcgtaacctc tctggtatgt gggcagagct accaggttaaggtcctccct agggtttgca 6600 aaaccctcat gggatcatga gccatacaga accgacctgtgtgtctccag agtctgtaat 6660 taacacaggc attttgagga aatgcgtggc ctcaggccccactcccggct acccccatcc 6720 cactatgcct agtatagtct agctgccctg gtacaattctcccagtatct tgcaggcccc 6780 tatttcctat tcctactctg ctcatctggc tctcaggaaccttcttggcc ttccctttca 6840 gacctctaca gatgccagcc ttgtgggaaa gaagagtgggcacctgaggg aagccagacc 6900 tgcttcccgc gcactgtggt gtttttggct ttgcgtgagcacacctcttg ggtgctgctg 6960 gcagctaaca cgctgctgct gctgctgctg cttgggactgctggcctgtt tgcctggcac 7020 ctagacaccc ctgtggtgag gtcagcaggg ggccgcctgtgctttcttat gctgggctcc 7080 ctggcagcag gtagtggcag cctctatggc ttctttggggaacccacaag gcctgcgtgc 7140 ttgctacgcc aggccctctt tgcccttggt ttcaccatcttcctgtcctg cctgacagtt 7200 cgctcattcc aactaatcat catcttcaag ttttccaccaaggtacctac attctaccac 7260 gcctgggtcc aaaaccacgg tgctggcctg tttgtgatgatcagctcagc ggcccagctg 7320 cttatctgtc taacttggct ggtggtgtgg accccactgcctgctaggga ataccagcgc 7380 ttcccccatc tggtgatgct tgagtgcaca gagaccaactccctgggctt catactggcc 7440 ttcctctaca atggcctcct ctccatcagt gcctttgcctgcagctacct gggtaaggac 7500 ttgccagaga actacaacga ggccaaatgt gtcaccttcagcctgctctt caacttcgtg 7560 tcctggatcg ccttcttcac cacggccagc gtctacgacggcaagtacct gcctgcggcc 7620 aacatgatgg ctgggctgag cagcctgagc agcggcttcggtgggtattt tctgcctaag 7680 tgctacgtga tcctctgccg cccagacctc aacagcacagagcacttcca ggcctccatt 7740 caggactaca cgaggcgctg cggctccacc tgaccagtgggtcagcaggc acggctggca 7800 gccttctctg ccctgagggt cgaaggtcga gcaggccgggggtgtccggg aggtctttgg 7860 gcatcgcggt ctggggttgg gacgtgtaag cgcctgggagagcctagacc aggctccggg 7920 ctgccaataa agaagtgaaa tgcgtatctg gtctcctgtcgtgggagagt gtgaggtgta 7980 acggattcaa gtctgaaccc agagcctgga aaaggctgaccgcccagatt gacgttgcta 8040 ggcaactccg gaggcgggcc cagcgccaaa agaacagggcgaggcgtcgt ccccgcatcc 8100 cattggccgt tctctgcggg gccccgccct cgggggccggagctagaagc tctacgcttc 8160 cgaggcgcac ctcctggcct gcacgctttg acgt 8194 162526 DNA Homo sapiens 16 atgctgctct gcacggctcg cctggtcggc ctgcagcttctcatttcctg ctgctgggcc 60 tttgcctgcc atagcacgga gtcttctcct gacttcaccctccccggaga ttacctcctg 120 gcaggcctgt tccctctcca ttctggctgt ctgcaggtgaggcacagacc cgaggtgacc 180 ctgtgtgaca ggtcttgtag cttcaatgag catggctaccacctcttcca ggctatgcgg 240 cttggggttg aggagataaa caactccacg gccctgctgcccaacatcac cctggggtac 300 cagctgtatg atgtgtgttc tgactctgcc aatgtgtatgccacgctgag agtgctctcc 360 ctgccagggc aacaccacat agagctccaa ggagaccttctccactattc ccctacggtg 420 ctggcagtga ttgggcctga cagcaccaac cgtgctgccaccacagccgc cctgctgagc 480 cctttcctgg tgcccatgat tagctatgcg gccagcagcgagacgctcag cgtgaagcgg 540 cagtatccct ctttcctgcg caccatcccc aatgacaagtaccaggtgga gaccatggtg 600 ctgctgctgc agaagttcgg gtggacctgg atctctctggttggcagcag tgacgactat 660 gggcagctag gggtgcaggc actggagaac caggccactggtcaggggat ctgcattgct 720 ttcaaggaca tcatgccctt ctctgcccag gtgggcgatgagaggatgca gtgcctcatg 780 cgccacctgg cccaggccgg ggccaccgtc gtggttgttttttccagccg gcagttggcc 840 agggtgtttt tcgagtccgt ggtgctgacc aacctgactggcaaggtgtg ggtcgcctca 900 gaagcctggg ccctctccag gcacatcact ggggtgcccgggatccagcg cattgggatg 960 gtgctgggcg tggccatcca gaagagggct gtccctggcctgaaggcgtt tgaagaagcc 1020 tatgcccggg cagacaagaa ggcccctagg ccttgccacaagggctcctg gtgcagcagc 1080 aatcagctct gcagagaatg ccaagctttc atggcacacacgatgcccaa gctcaaagcc 1140 ttctccatga gttctgccta caacgcatac cgggctgtgtatgcggtggc ccatggcctc 1200 caccagctcc tgggctgtgc ctctggagct tgttccaggggccgagtcta cccctggcag 1260 cttttggagc agatccacaa ggtgcatttc cttctacacaaggacactgt ggcgtttaat 1320 gacaacagag atcccctcag tagctataac ataattgcctgggactggaa tggacccaag 1380 tggaccttca cggtcctcgg ttcctccaca tggtctccagttcagctaaa cataaatgag 1440 accaaaatcc agtggcacgg aaaggacaac caggtgcctaagtctgtgtg ttccagcgac 1500 tgtcttgaag ggcaccagcg agtggttacg ggtttccatcactgctgctt tgagtgtgtg 1560 ccctgtgggg ctgggacctt cctcaacaag agtgacctctacagatgcca gccttgtggg 1620 aaagaagagt gggcacctga gggaagccag acctgcttcccgcgcactgt ggtgtttttg 1680 gctttgcgtg agcacacctc ttgggtgctg ctggcagctaacacgctgct gctgctgctg 1740 ctgcttggga ctgctggcct gtttgcctgg cacctagacacccctgtggt gaggtcagca 1800 gggggccgcc tgtgctttct tatgctgggc tccctggcagcaggtagtgg cagcctctat 1860 ggcttctttg gggaacccac aaggcctgcg tgcttgctacgccaggccct ctttgccctt 1920 ggtttcacca tcttcctgtc ctgcctgaca gttcgctcattccaactaat catcatcttc 1980 aagttttcca ccaaggtacc tacattctac cacgcctgggtccaaaacca cggtgctggc 2040 ctgtttgtga tgatcagctc agcggcccag ctgcttatctgtctaacttg gctggtggtg 2100 tggaccccac tgcctgctag ggaataccag cgcttcccccatctggtgat gcttgagtgc 2160 acagagacca actccctggg cttcatactg gccttcctctacaatggcct cctctccatc 2220 agtgcctttg cctgcagcta cctgggtaag gacttgccagagaactacaa cgaggccaaa 2280 tgtgtcacct tcagcctgct cttcaacttc gtgtcctggatcgccttctt caccacggcc 2340 agcgtctacg acggcaagta cctgcctgcg gccaacatgatggctgggct gagcagcctg 2400 agcagcggct tcggtgggta ttttctgcct aagtgctacgtgatcctctg ccgcccagac 2460 ctcaacagca cagagcactt ccaggcctcc attcaggactacacgaggcg ctgcggctcc 2520 acctga 2526 17 841 PRT Homo sapiens 17 MetLeu Leu Cys Thr Ala Arg Leu Val Gly Leu Gln Leu Leu Ile Ser 1 5 10 15Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe 20 25 30Thr Leu Pro Gly Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser 35 40 45Gly Cys Leu Gln Val Arg His Arg Pro Glu Val Thr Leu Cys Asp Arg 50 55 60Ser Cys Ser Phe Asn Glu His Gly Tyr His Leu Phe Gln Ala Met Arg 65 70 7580 Leu Gly Val Glu Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn Ile 85 9095 Thr Leu Gly Tyr Gln Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val 100105 110 Tyr Ala Thr Leu Arg Val Leu Ser Leu Pro Gly Gln His His Ile Glu115 120 125 Leu Gln Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala ValIle 130 135 140 Gly Pro Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala LeuLeu Ser 145 150 155 160 Pro Phe Leu Val Pro Met Ile Ser Tyr Ala Ala SerSer Glu Thr Leu 165 170 175 Ser Val Lys Arg Gln Tyr Pro Ser Phe Leu ArgThr Ile Pro Asn Asp 180 185 190 Lys Tyr Gln Val Glu Thr Met Val Leu LeuLeu Gln Lys Phe Gly Trp 195 200 205 Thr Trp Ile Ser Leu Val Gly Ser SerAsp Asp Tyr Gly Gln Leu Gly 210 215 220 Val Gln Ala Leu Glu Asn Gln AlaThr Gly Gln Gly Ile Cys Ile Ala 225 230 235 240 Phe Lys Asp Ile Met ProPhe Ser Ala Gln Val Gly Asp Glu Arg Met 245 250 255 Gln Cys Leu Met ArgHis Leu Ala Gln Ala Gly Ala Thr Val Val Val 260 265 270 Val Phe Ser SerArg Gln Leu Ala Arg Val Phe Phe Glu Ser Val Val 275 280 285 Leu Thr AsnLeu Thr Gly Lys Val Trp Val Ala Ser Glu Ala Trp Ala 290 295 300 Leu SerArg His Ile Thr Gly Val Pro Gly Ile Gln Arg Ile Gly Met 305 310 315 320Val Leu Gly Val Ala Ile Gln Lys Arg Ala Val Pro Gly Leu Lys Ala 325 330335 Phe Glu Glu Ala Tyr Ala Arg Ala Asp Lys Lys Ala Pro Arg Pro Cys 340345 350 His Lys Gly Ser Trp Cys Ser Ser Asn Gln Leu Cys Arg Glu Cys Gln355 360 365 Ala Phe Met Ala His Thr Met Pro Lys Leu Lys Ala Phe Ser MetSer 370 375 380 Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala HisGly Leu 385 390 395 400 His Gln Leu Leu Gly Cys Ala Ser Gly Ala Cys SerArg Gly Arg Val 405 410 415 Tyr Pro Trp Gln Leu Leu Glu Gln Ile His LysVal His Phe Leu Leu 420 425 430 His Lys Asp Thr Val Ala Phe Asn Asp AsnArg Asp Pro Leu Ser Ser 435 440 445 Tyr Asn Ile Ile Ala Trp Asp Trp AsnGly Pro Lys Trp Thr Phe Thr 450 455 460 Val Leu Gly Ser Ser Thr Trp SerPro Val Gln Leu Asn Ile Asn Glu 465 470 475 480 Thr Lys Ile Gln Trp HisGly Lys Asp Asn Gln Val Pro Lys Ser Val 485 490 495 Cys Ser Ser Asp CysLeu Glu Gly His Gln Arg Val Val Thr Gly Phe 500 505 510 His His Cys CysPhe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu 515 520 525 Asn Lys SerAsp Leu Tyr Arg Cys Gln Pro Cys Gly Lys Glu Glu Trp 530 535 540 Ala ProGlu Gly Ser Gln Thr Cys Phe Pro Arg Thr Val Val Phe Leu 545 550 555 560Ala Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu 565 570575 Leu Leu Leu Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu 580585 590 Asp Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met595 600 605 Leu Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe PheGly 610 615 620 Glu Pro Thr Arg Pro Ala Cys Leu Leu Arg Gln Ala Leu PheAla Leu 625 630 635 640 Gly Phe Thr Ile Phe Leu Ser Cys Leu Thr Val ArgSer Phe Gln Leu 645 650 655 Ile Ile Ile Phe Lys Phe Ser Thr Lys Val ProThr Phe Tyr His Ala 660 665 670 Trp Val Gln Asn His Gly Ala Gly Leu PheVal Met Ile Ser Ser Ala 675 680 685 Ala Gln Leu Leu Ile Cys Leu Thr TrpLeu Val Val Trp Thr Pro Leu 690 695 700 Pro Ala Arg Glu Tyr Gln Arg PhePro His Leu Val Met Leu Glu Cys 705 710 715 720 Thr Glu Thr Asn Ser LeuGly Phe Ile Leu Ala Phe Leu Tyr Asn Gly 725 730 735 Leu Leu Ser Ile SerAla Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu 740 745 750 Pro Glu Asn TyrAsn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe 755 760 765 Asn Phe ValSer Trp Ile Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp 770 775 780 Gly LysTyr Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu 785 790 795 800Ser Ser Gly Phe Gly Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile Leu 805 810815 Cys Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gln Ala Ser Ile Gln 820825 830 Asp Tyr Thr Arg Arg Cys Gly Ser Thr 835 840 18 14 PRT ArtificialSequence MOD_RES (1) Thr or Arg 18 Xaa Cys Xaa Xaa Arg Xaa Xaa Xaa PheLeu Xaa Xaa Xaa Glu 1 5 10 19 15 PRT Artificial Sequence MOD_RES (1) Leuor Gln 19 Xaa Pro Xaa Xaa Tyr Asn Xaa Ala Xaa Xaa Xaa Thr Xaa Xaa Xaa 15 10 15 20 3563 DNA Homo sapiens 20 agcctggcag tggcctcagg cagagtctgacgcgcacaaa ctttcaggcc caggaagcga 60 ggacaccact ggggccccag ggtgtggcaagtgaggatgg caagggtttt gctaaacaaa 120 tcctctgccc gctccccgcc ccgggctcactccatgtgag gccccagtcg gggcagccac 180 ctgccgtgcc tgttggaagt tgcctctgccatgctgggcc ctgctgtcct gggcctcagc 240 ctctgggctc tcctgcaccc tgggacgggggccccattgt gcctgtcaca gcaacttagg 300 atgaaggggg actacgtgct gggggggctgttccccctgg gcgaggccga ggaggctggc 360 ctccgcagcc ggacacggcc cagcagccctgtgtgcacca ggtacagagg tgggacggcc 420 tgggtcgggg tcagggtgac caggtctggggtgctcctga gctggggccg aggtggccat 480 ctgcggttct gtgtggcccc aggttctcctcaaacggcct gctctgggca ctggccatga 540 aaatggccgt ggaggagatc aacaacaagtcggatctgct gcccgggctg cgcctgggct 600 acgacctctt tgatacgtgc tcggagcctgtggtggccat gaagcccagc ctcatgttcc 660 tggccaaggc aggcagccgc gacatcgccgcctactgcaa ctacacgcag taccagcccc 720 gtgtgctggc tgtcatcggg ccccactcgtcagagctcgc catggtcacc ggcaagttct 780 tcagcttctt cctcatgccc cagtggggcgccccccacca tcacccaccc ccaaccaacc 840 cctgccccgt gggagcccct tgtgtcaggagaatgctaca tgcaccccac ccagccctgc 900 cctgggagcc ctgtgtcaga agatgctcttggccttgcag gtcagctacg gtgctagcat 960 ggagctgctg agcgcccggg agaccttcccctccttcttc cgcaccgtgc ccagcgaccg 1020 tgtgcagctg acggccgccg cggagctgctgcaggagttc ggctggaact gggtggccgc 1080 cctgggcagc gacgacgagt acggccggcagggcctgagc atcttctcgg ccctggccgc 1140 ggcacgcggc atctgcatcg cgcacgagggcctggtgccg ctgccccgtg ccgatgactc 1200 gcggctgggg aaggtgcagg acgtcctgcaccaggtgaac cagagcagcg tgcaggtggt 1260 gctgctgttc gcctccgtgc acgccgcccacgccctcttc aactacagca tcagcagcag 1320 gctctcgccc aaggtgtggg tggccagcgaggcctggctg acctctgacc tggtcatggg 1380 gctgcccggc atggcccaga tgggcacggtgcttggcttc ctccagaggg gtgcccagct 1440 gcacgagttc ccccagtacg tgaagacgcacctggccctg gccaccgacc cggccttctg 1500 ctctgccctg ggcgagaggg agcagggtctggaggaggac gtggtgggcc agcgctgccc 1560 gcagtgtgac tgcatcacgc tgcagaacgtgagcgcaggg ctaaatcacc accagacgtt 1620 ctctgtctac gcagctgtgt atagcgtggcccaggccctg cacaacactc ttcagtgcaa 1680 cgcctcaggc tgccccgcgc aggaccccgtgaagccctgg caggtgagcc cgggagatgg 1740 gggtgtgctg tcctctgcat gtgcccaggccaccaggcac ggccaccacg cctgagctgg 1800 aggtggctgg cggctcagcc ccgtcccccgcccgcagctc ctggagaaca tgtacaacct 1860 gaccttccac gtgggcgggc tgccgctgcggttcgacagc agcggaaacg tggacatgga 1920 gtacgacctg aagctgtggg tgtggcagggctcagtgccc aggctccacg acgtgggcag 1980 gttcaacggc agcctcagga cagagcgcctgaagatccgc tggcacacgt ctgacaacca 2040 ggtgaggtga gggtgggtgt gccaggcgtgcccgtggtag cccccgcggc agggcgcagc 2100 ctgggggtgg gggccgttcc agtctcccgtgggcatgccc agccgagcag agccagaccc 2160 caggcctgtg cgcagaagcc cgtgtcccggtgctcgcggc agtgccagga gggccaggtg 2220 cgccgggtca aggggttcca ctcctgctgctacgactgtg tggactgcga ggcgggcagc 2280 taccggcaaa acccaggtga gccgccttcccggcaggcgg gggtgggaac gcagcagggg 2340 agggtcctgc caagtcctga ctctgagaccagagcccaca gggtacaaga cgaacaccca 2400 gcgcccttct cctctctcac agacgacatcgcctgcacct tttgtggcca ggatgagtgg 2460 tccccggagc gaagcacacg ctgcttccgccgcaggtctc ggttcctggc atggggcgag 2520 ccggctgtgc tgctgctgct cctgctgctgagcctggcgc tgggccttgt gctggctgct 2580 ttggggctgt tcgttcacca tcgggacagcccactggttc aggcctcggg ggggcccctg 2640 gcctgctttg gcctggtgtg cctgggcctggtctgcctca gcgtcctcct gttccctggc 2700 cagcccagcc ctgcccgatg cctggcccagcagcccttgt cccacctccc gctcacgggc 2760 tgcctgagca cactcttcct gcaggcggccgagatcttcg tggagtcaga actgcctctg 2820 agctgggcag accggctgag tggctgcctgcgggggccct gggcctggct ggtggtgctg 2880 ctggccatgc tggtggaggt cgcactgtgcacctggtacc tggtggcctt cccgccggag 2940 gtggtgacgg actggcacat gctgcccacggaggcgctgg tgcactgccg cacacgctcc 3000 tgggtcagct tcggcctagc gcacgccaccaatgccacgc tggcctttct ctgcttcctg 3060 ggcactttcc tggtgcggag ccagccgggctgctacaacc gtgcccgtgg cctcaccttt 3120 gccatgctgg cctacttcat cacctgggtctcctttgtgc ccctcctggc caatgtgcag 3180 gtggtcctca ggcccgccgt gcagatgggcgccctcctgc tctgtgtcct gggcatcctg 3240 gctgccttcc acctgcccag gtgttacctgctcatgcggc agccagggct caacaccccc 3300 gagttcttcc tgggaggggg ccctggggatgcccaaggcc agaatgacgg gaacacagga 3360 aatcagggga aacatgagtg acccaaccctgtgatctcag ccccggtgaa cccagactta 3420 gctgcgatcc cccccaagcc agcaatgacccgtgtctcgc tacagagacc ctcccgctct 3480 aggttctgac cccaggttgt ctcctgaccctgaccccaca gtgagcccta ggcctggagc 3540 acgtggacac ccctgtgacc atc 3563

What is claimed:
 1. An isolated nucleic acid selected from the groupconsisting of: (i) a genomic DNA sequence consisting essentially of anucleic acid sequence coding for a T1R mammalian G protein-coupledreceptor polypeptide active in taste signaling, wherein said nucleicacid sequence consists essentially of a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs 15 and 20; (ii) a genomic DNAsequence consisting essentially of a nucleic acid sequence coding for aT1R mammalian G protein-coupled receptor polypeptide have an amino acidsequence selected from the group consisting of SEQ ID NOs 4, 14, and 17;(iii) a genomic DNA sequence having at least about 50% identity to anucleic acid sequence coding for a T1R mammalian G protein-coupledreceptor polypeptide, wherein said nucleic acid sequence consistsessentially of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs 15 and 20; a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs 15 and 20 (iv) a genomic DNAsequence consisting essentially of a nucleic acid sequence coding for aT1R mammalian G protein-coupled receptor polypeptide having an aminoacid sequence that is at least about 40% identical to the amino acidsequence selected from the group consisting of SEQ ID NOs 4, 14, and 17;(v) a genomic DNA sequence consisting essentially of a sequence codingfor a T1R mammalian G protein-coupled receptor polypeptide comprising aconsensus sequence selected from the group consisting of SEQ ID NOs 18and 19, and sequences having at least about 75% identity to SEQ ID NOs18 or 19; (vi) a cDNA sequence having the same nucleic acid sequence asthe T1R mammalian G protein-coupled receptor polypeptide coding regionin the genomic DNA sequence selected from the group consisting of SEQ IDNOs 15 and 20; (vii) a cDNA sequence coding for a T1R mammalian Gprotein-coupled receptor polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs 4, 14, and 17; (viii) acDNA sequence coding for a T1R mammalian G protein-coupled receptorpolypeptide comprising a consensus sequence selected from the groupconsisting of SEQ ID NOs 18 and 19, and sequences having at least about75% identity to SEQ ID NOs 18 or 19; (ix) a cDNA sequence selected fromthe group consisting of SEQ ID NOs 3, 13 and 16; (x) a cDNA sequencehaving at least about 50% sequence identity to the T1R G protein-coupledreceptor polypeptide coding region in the genomic DNA sequence selectedfrom the group consisting of SEQ ID NOs 15 and 20; (xi) a cDNA sequencehaving at least about 50% sequence identity to a sequence encoding a T1Rmammalian G protein-coupled receptor polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NOs 4, 14, and 17;(xii) a cDNA sequence having at least about 50% identity to a sequenceselected from the group consisting of SEQ ID NOs 3, 13, and 16; (xiii) avariant of a nucleotide sequence selected from the group consisting ofSEQ ID NOs 3, 13, 15, 16, and 20, containing at least one conservativesubstitution in a region coding for a T1R G protein-coupled receptorpolypeptide active in taste signaling; (xiv) a variant of a nucleotidesequence encoding a T1R G protein-coupled receptor polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NOs 4,14, and 17, containing at least one conservative substitution in a T1Rmammalian G protein-coupled receptor polypeptide coding region; and (xv)a variant of a cDNA sequence selected from the group consisting of SEQID NOs 3, 13, and 16, containing at least one conservative substitution.2. An isolated genomic DNA molecule consisting essentially of a nucleicacid sequence coding for a mammalian G protein-coupled receptorpolypeptide, wherein said nucleic acid sequence consists essential ofSEQ ID NO 15 or
 20. 3. An isolated RNA molecule transcribed from theisolated DNA molecule of claim
 2. 4. An isolated nucleic acid moleculethat hybridizes to the DNA molecule of claim 2 under stringenthybridization conditions.
 5. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 2 under moderate hybridizationconditions.
 6. An isolated fragment of the genomic DNA molecule of claim2 that is at least about 20 to 30 nucleotide bases in length.
 7. Achimeric or fused nucleic acid molecule, wherein said chimeric or fusednucleic acid molecule comprises at least part of the coding sequencecontained in the DNA molecule of claim 2, and at least part of aheterologous coding sequence, wherein transcription of said chimeric orfused nucleic acid molecule results in a single chimeric nucleic acidtranscript.
 8. The chimeric or fused nucleic acid molecule of claim 7,wherein said heterologous coding sequence is from a sequence encoding adifferent G protein-coupled receptor.
 9. The chimeric or fused nucleicacid molecule of claim 7, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 10. The chimeric or fusednucleic acid molecule of claim 9, wherein said heterologous codingsequence is from a mammalian rhodopsin gene.
 11. The chimeric or fusednucleic acid molecule of claim 7, wherein said heterologous codingsequence is from a gene encoding green fluorescent protein or otherdetectable marker gene.
 12. An isolated genomic DNA molecule consistingessentially of a nucleic acid sequence coding for a mammalian Gprotein-coupled receptor having an amino acid sequence selected from thegroup consisting of SEQ ID NOs 4, 14, and
 17. 13. An isolated RNAmolecule transcribed from the isolated DNA molecule of claim
 12. 14. Anisolated nucleic acid molecule that hybridizes to the DNA molecule ofclaim 12 under stringent hybridization conditions.
 15. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 12under moderate hybridization conditions.
 16. An isolated fragment of thegenomic DNA molecule of claim 12 that is at least about 20 to 30nucleotide bases in length.
 17. A chimeric or fused nucleic acidmolecule, wherein said chimeric or fused nucleic acid molecule comprisesat least part of the coding sequence contained in the DNA molecule ofclaim 12, and at least part of a heterologous coding sequence, whereintranscription of said chimeric or fused nucleic acid molecule results ina single chimeric nucleic acid transcript.
 18. The chimeric or fusednucleic acid molecule of claim 17, wherein said heterologous codingsequence is from a sequence encoding a different G protein-coupledreceptor.
 19. The chimeric or fused nucleic acid molecule of claim 17,wherein said heterologous coding sequence is a sequence that facilitatesexpression of said mammalian G protein-coupled receptor polypeptide onthe surface of a cell.
 20. The chimeric or fused nucleic acid moleculeof claim 19, wherein said heterologous coding sequence is from amammalian rhodopsin gene.
 21. The chimeric or fused nucleic acidmolecule of claim 17, wherein said heterologous coding sequence is froma gene encoding green fluorescent protein or other detectable markergene.
 22. An isolated genomic DNA molecule consisting essentially of anucleic acid sequence having at least about 50% identity to a nucleicacid sequence coding for a mammalian G protein-coupled receptorpolypeptide, wherein said nucleic acid sequence consists essentially ofa nucleic acid sequence selected from the group consisting of SEQ ID NOs15 and
 20. 23. An isolated RNA molecule transcribed from the isolatedDNA molecule of claim
 22. 24. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 22 under stringent hybridizationconditions.
 25. An isolated nucleic acid molecule that hybridizes to theDNA molecule of claim 22 under moderate hybridization conditions.
 26. Anisolated fragment of the genomic DNA molecule of claim 22 that is atleast about 20 to 30 nucleotide bases in length.
 27. A chimeric or fusednucleic acid molecule, wherein said chimeric or fused nucleic acidmolecule comprises at least part of the coding sequence contained in theDNA molecule of claim 22, and at least part of a heterologous codingsequence, wherein transcription of said chimeric or fused nucleic acidmolecule results in a single chimeric nucleic acid transcript.
 28. Thechimeric or fused nucleic acid molecule of claim 27, wherein saidheterologous coding sequence is from a sequence encoding a different Gprotein-coupled receptor.
 29. The chimeric or fused nucleic acidmolecule of claim 27, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 30. The chimeric or fusednucleic acid molecule of claim 29, wherein said heterologous codingsequence is from a mammalian rhodopsin gene.
 31. The chimeric or fusednucleic acid molecule of claim 27, wherein said heterologous codingsequence is from a gene encoding green fluorescent protein or otherdetectable marker gene.
 32. An isolated genomic DNA molecule consistingessentially of a nucleic acid sequence coding for a mammalian Gprotein-coupled receptor having an amino acid sequence that is at leastabout 40% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 14, and
 17. 33. An isolated RNA moleculetranscribed from the isolated DNA molecule of claim
 32. 34. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 32under stringent hybridization conditions.
 35. An isolated nucleic acidmolecule that hybridizes to the DNA molecule of claim 32 under moderatehybridization conditions.
 36. An isolated fragment of the genomic DNAmolecule of claim 32 that is at least about 20 to 30 nucleotide bases inlength.
 37. A chimeric or fused nucleic acid molecule, wherein saidchimeric or fused nucleic acid molecule comprises at least part of thecoding sequence contained in the DNA molecule of claim 32, and at leastpart of a heterologous coding sequence, wherein transcription of saidchimeric or fused nucleic acid molecule results in a single chimericnucleic acid transcript.
 38. The chimeric or fused nucleic acid moleculeof claim 37, wherein said heterologous coding sequence is from asequence encoding a different G protein-coupled receptor.
 39. Thechimeric or fused nucleic acid molecule of claim 37, wherein saidheterologous coding sequence is a sequence that facilitates expressionof said mammalian G protein-coupled receptor polypeptide on the surfaceof a cell.
 40. The chimeric or fused nucleic acid molecule of claim 39,wherein said heterologous coding sequence is from a mammalian rhodopsingene.
 41. The chimeric or fused nucleic acid molecule of claim 37,wherein said heterologous coding sequence is from a gene encoding greenfluorescent protein or other detectable marker gene.
 42. An isolatedcDNA molecule comprising a nucleic acid sequence having the samesequence as the mammalian G protein-coupled receptor polypeptide codingregion contained in a genomic DNA sequence consisting essentially of anucleic acid sequence selected from the group consisting of SEQ ID NOs15 and
 20. 43. An isolated RNA molecule transcribed from the isolatedDNA molecule of claim
 42. 44. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 42 under stringent hybridizationconditions.
 45. An isolated nucleic acid molecule that hybridizes to theDNA molecule of claim 42 under moderate hybridization conditions.
 46. Anisolated fragment of the genomic DNA molecule of claim 42 that is atleast about 20 to 30 nucleotide bases in length.
 47. A chimeric or fusednucleic acid molecule, wherein said chimeric or fused nucleic acidmolecule comprises at least part of the coding sequence contained in theDNA molecule of claim 42, and at least part of a heterologous codingsequence, wherein transcription of said chimeric or fused nucleic acidmolecule results in a single chimeric nucleic acid transcript.
 48. Thechimeric or fused nucleic acid molecule of claim 47, wherein saidheterologous coding sequence is from a sequence encoding a different Gprotein-coupled receptor.
 49. The chimeric or fused nucleic acidmolecule of claim 47, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 50. The chimeric or fusednucleic acid molecule of claim 49, wherein said heterologous codingsequence is from a mammalian rhodopsin gene.
 51. The chimeric or fusednucleic acid molecule of claim 47, wherein said heterologous codingsequence is from a gene encoding green fluorescent protein or otherdetectable marker gene.
 52. A nucleic acid molecule comprising theisolated cDNA of claim 42 operably linked to a heterologous promoterthat is either regulatable or constitutive.
 53. The nucleic acidmolecule of claim 52, wherein said regulatable promoter is inducibleunder specific environmental or developmental conditions.
 54. Anisolated cDNA molecule comprising a nucleic acid sequence coding for a Gprotein-coupled receptor polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NOs 4, 14, and
 17. 55. Anisolated RNA molecule transcribed from the isolated DNA molecule ofclaim
 54. 56. An isolated nucleic acid molecule that hybridizes to theDNA molecule of claim 54 under stringent hybridization conditions. 57.An isolated nucleic acid molecule that hybridizes to the DNA molecule ofclaim 54 under moderate hybridization conditions.
 58. An isolatedfragment of the genomic DNA molecule of claim 54 that is at least about20 to 30 nucleotide bases in length.
 59. A chimeric or fused nucleicacid molecule, wherein said chimeric or fused nucleic acid moleculecomprises at least part of the coding sequence contained in the DNAmolecule of claim 54, and at least part of a heterologous codingsequence, wherein transcription of said chimeric or fused nucleic acidmolecule results in a single chimeric nucleic acid transcript.
 60. Thechimeric or fused nucleic acid molecule of claim 59, wherein saidheterologous coding sequence is from a sequence encoding a different Gprotein-coupled receptor.
 61. The chimeric or fused nucleic acidmolecule of claim 59, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 62. The chimeric or fusednucleic acid molecule of claim 61, wherein said heterologous codingsequence is from a mammalian rhodopsin gene.
 63. The chimeric or fusednucleic acid molecule of claim 59, wherein said heterologous codingsequence is from a gene encoding green fluorescent protein or otherdetectable marker gene.
 64. A nucleic acid molecule comprising theisolated cDNA of claim 54 operably linked to a heterologous promoterthat is either regulatable or constitutive.
 65. The nucleic acidmolecule of claim 64, wherein said regulatable promoter is inducibleunder specific environmental or developmental conditions.
 66. Anisolated cDNA molecule comprising a nucleic acid sequence having atleast about 50% sequence identity to the G protein-coupled receptorpolypeptide coding region in a genomic DNA sequence consistingessentially of a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs 15 and
 20. 67. An isolated RNA moleculetranscribed from the isolated DNA molecule of claim
 66. 68. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 66under stringent hybridization conditions.
 69. An isolated nucleic acidmolecule that hybridizes to the DNA molecule of claim 66 under moderatehybridization conditions.
 70. An isolated fragment of the genomic DNAmolecule of claim 66 that is at least about 20 to 30 nucleotide bases inlength.
 71. A chimeric or fused nucleic acid molecule, wherein saidchimeric or fused nucleic acid molecule comprises at least part of thecoding sequence contained in the DNA molecule of claim 66, and at leastpart of a heterologous coding sequence, wherein transcription of saidchimeric or fused nucleic acid molecule results in a single chimericnucleic acid transcript.
 72. The chimeric or fused nucleic acid moleculeof claim 71, wherein said heterologous coding sequence is from asequence encoding a different G protein-coupled receptor.
 73. Thechimeric or fused nucleic acid molecule of claim 71, wherein saidheterologous coding sequence is a sequence that facilitates expressionof said mammalian G protein-coupled receptor polypeptide on the surfaceof a cell.
 74. The chimeric or fused nucleic acid molecule of claim 73,wherein said heterologous coding sequence is from a mammalian rhodopsingene.
 75. The chimeric or fused nucleic acid molecule of claim 71,wherein said heterologous coding sequence is from a gene encoding greenfluorescent protein or other detectable marker gene.
 76. A nucleic acidmolecule comprising the isolated cDNA of claim 66 operably linked to aheterologous promoter that is either regulatable or constitutive. 77.The nucleic acid molecule of claim 76, wherein said regulatable promoteris inducible under specific environmental or developmental conditions.78. An isolated cDNA molecule comprising a nucleic acid sequence havingat least about 40% sequence identity to a sequence encoding a mammalianT1R G protein-coupled receptor polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs 4, 10, 12, 14,and
 17. 79. An isolated RNA molecule transcribed from the isolated DNAmolecule of claim
 78. 80. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 78 under stringent hybridizationconditions.
 81. An isolated nucleic acid molecule that hybridizes to theDNA molecule of claim 78 under moderate hybridization conditions.
 82. Anisolated fragment of the genomic DNA molecule of claim 78 that is atleast about 20 to 30 nucleotide bases in length.
 83. A chimeric or fusednucleic acid molecule, wherein said chimeric or fused nucleic acidmolecule comprises at least part of the coding sequence contained in theDNA molecule of claim 78, and at least part of a heterologous codingsequence, wherein transcription of said chimeric or fused nucleic acidmolecule results in a single chimeric nucleic acid transcript.
 84. Thechimeric or fused nucleic acid molecule of claim 83, wherein saidheterologous coding sequence is from a sequence encoding a different Gprotein-coupled receptor.
 85. The chimeric or fused nucleic acidmolecule of claim 83, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 86. The chimeric or fusednucleic acid molecule of claim 85, wherein said heterologous codingsequence is from a mammalian rhodopsin gene.
 87. The chimeric or fusednucleic acid molecule of claim 83, wherein said heterologous codingsequence is from a gene encoding green fluorescent protein or otherdetectable marker gene.
 88. A nucleic acid molecule comprising theisolated cDNA of claim 78 operably linked to a heterologous promoterthat is either regulatable or constitutive.
 89. The nucleic acidmolecule of claim 88, wherein said regulatable promoter is inducibleunder specific environmental or developmental conditions.
 90. Anisolated variant DNA molecule comprising a nucleotide sequenceconsisting essentially of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs 15 and 20, containing at least oneconservative substitution in a region coding for a G protein-coupledreceptor active in taste signaling.
 91. An isolated RNA moleculetranscribed from the isolated DNA molecule of claim
 90. 92. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 90under stringent hybridization conditions.
 93. An isolated nucleic acidmolecule that hybridizes to the DNA molecule of claim 90 under moderatehybridization conditions.
 94. An isolated fragment of the genomic DNAmolecule of claim 90 that is at least about 20 to 30 nucleotide bases inlength.
 95. A chimeric or fused nucleic acid molecule, wherein saidchimeric or fused nucleic acid molecule comprises at least part of thecoding sequence contained in the DNA molecule of claim 90, and at leastpart of a heterologous coding sequence, wherein transcription of saidchimeric or fused nucleic acid molecule results in a single chimericnucleic acid transcript.
 96. The chimeric or fused nucleic acid moleculeof claim 95, wherein said heterologous coding sequence is from asequence encoding a different G protein-coupled receptor.
 97. Thechimeric or fused nucleic acid molecule of claim 95, wherein saidheterologous coding sequence is a sequence that facilitates expressionof said mammalian G protein-coupled receptor polypeptide on the surfaceof a cell.
 98. The chimeric or fused nucleic acid molecule of claim 97,wherein said heterologous coding sequence is from a mammalian rhodopsingene.
 99. The chimeric or fused nucleic acid molecule of claim 95,wherein said heterologous coding sequence is from a gene encoding greenfluorescent protein or other detectable marker gene.
 100. A cDNAmolecule having the same nucleic acid sequence as the coding region ofthe variant DNA molecule of claim
 90. 101. A nucleic acid moleculecomprising the cDNA of claim 100 operably linked to a heterologouspromoter that is either regulatable or constitutive.
 102. The nucleicacid molecule of claim 101, wherein said regulatable promoter isinducible under specific environmental or developmental conditions. 103.An isolated variant molecule comprising a nucleotide sequence encoding apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs 4, 14, and 17, containing at least oneconservative substitution in a coding region.
 104. An isolated RNAmolecule transcribed from the isolated DNA molecule of claim
 103. 105.An isolated nucleic acid molecule that hybridizes to the DNA molecule ofclaim 103 under stringent hybridization conditions.
 106. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 103under moderate hybridization conditions.
 107. An isolated fragment ofthe genomic DNA molecule of claim 103 that is at least about 20 to 30nucleotide bases in length.
 108. A chimeric or fused nucleic acidmolecule, wherein said chimeric or fused nucleic acid molecule comprisesat least part of the coding sequence contained in the DNA molecule ofclaim 103, and at least part of a heterologous coding sequence, whereintranscription of said chimeric or fused nucleic acid molecule results ina single chimeric nucleic acid transcript.
 109. The chimeric or fusednucleic acid molecule of claim 108, wherein said heterologous codingsequence is from a sequence encoding a different G protein-coupledreceptor.
 110. The chimeric or fused nucleic acid molecule of claim 108,wherein said heterologous coding sequence is a sequence that facilitatesexpression of said mammalian G protein-coupled receptor polypeptide onthe surface of a cell.
 111. The chimeric or fused nucleic acid moleculeof claim 110, wherein said heterologous coding sequence is from amammalian rhodopsin gene.
 112. The chimeric or fused nucleic acidmolecule of claim 108, wherein said heterologous coding sequence is froma gene encoding green fluorescent protein or other detectable markergene.
 113. A cDNA molecule having the same nucleic acid sequence as thecoding region of the variant DNA molecule of claim
 103. 114. A nucleicacid molecule comprising the cDNA molecule of claim 113 operably linkedto a heterologous promoter that is either regulatable or constitutive.115. The nucleic acid molecule of claim 114, wherein said regulatablepromoter is inducible under specific environmental or developmentalconditions.
 116. The isolated nucleic acid molecule of claim 1, whereinsaid nucleic acid encodes a G protein-coupled receptor polypeptide thatis active in taste signaling in rat, mouse, or human.
 117. An expressionvector comprising an isolated nucleic acid molecule of claim 1, whereinsaid vector is selected from the group consisting of mammalian vectors,bacterial plasmids, bacterial phagemids, mammalian viruses andretroviruses, bacteriophage vectors and linear or circular DNA moleculescapable of integrating into a host cell genome.
 118. A host celltransfected with at least one of the expression vectors of claim 117,wherein said host cell expresses the encoded G protein-coupled receptorpolypeptides on the surface of said host cell.
 119. A nucleic acid arraycomprising at least about 20 to 30 nucleotides of at least one of theisolated nucleic acid molecules of claim 1, wherein the at least onenucleic acid molecules are linked covalently or noncovalently to a solidphase support.
 120. A method of screening for compounds that activatetaste signaling comprising: (i) contacting the host cell of claim 118with a putative taste activating compound; and (ii) measuring activityfrom said G protein-coupled receptor polypeptide expressed on the cellsurface.
 121. The method of claim 120, wherein said G protein-coupledreceptor polypeptide activity is measured by assayed by measuringchanges in intracellular Ca²⁺ levels, cAMP, cGMP and IP3, or G proteinbinding of GTPγS.
 122. The method of claim 120, wherein said host cellis transfected with at least one additional nucleic acid constructencoding a gene involved in taste signaling.
 123. The method of claim122, wherein said at least one additional gene encodes a G proteininvolved in taste signal transduction.
 124. The method of claim 123,wherein said G protein is a promiscuous G protein.
 125. A method ofscreening for compounds that modulate taste signaling transductioncomprising: (i) contacting a host cell according to claim 118 with aknown taste activating compound and a compound putatively involved intaste transduction modulation; (ii) contacting a host cell according toclaim 118 with a known taste activating compound alone; and (iii)comparing the activity from said G protein-coupled receptor polypeptideexpressed on the cell surface of the host cell of step (i) with theactivity from said G protein-coupled receptor polypeptide expressed onthe cell surface of the host cell of step (ii) to identify modulators oftaste transduction.
 126. The method of claim 125, wherein saidmodulatory compounds are selected from the group consisting ofactivators, inhibitors, stimulators, enhancers, agonists andantagonists.
 127. The method of claim 125, wherein said Gprotein-coupled receptor polypeptide activity is measured by assayed bymeasuring changes in intracellular Ca²⁺ levels, cAMP, cGMP and IP3, or Gprotein binding of GTPγS.
 128. The method of claim 125, wherein saidhost cell is transfected with at least one additional nucleic acidconstruct encoding a gene involved in taste signaling.
 129. The methodof claim 128, wherein said at least one additional gene encodes a Gprotein involved in taste signal transduction.
 130. The method of claim129, wherein said G protein is a promiscuous G protein.
 131. A method ofdetecting expression of a G protein-coupled receptor polypeptide gene ina cell comprising: (i) contacting said cell with a nucleic acid moleculethat hybridizes to the isolated nucleic acid molecule of claim 1 understringent conditions; and (ii) detecting hybridization in order todetect expression of said G protein-coupled receptor polypeptide gene.132. An isolated nucleic acid molecule encoding a G protein-coupledreceptor polypeptide active in taste signaling having the nucleotidesequence of SEQ ID NO:
 1. 133. An isolated nucleic acid moleculeencoding a G protein-coupled receptor polypeptide active in tastesignaling having the nucleotide sequence of SEQ ID NO:
 2. 134. Anisolated nucleic acid molecule encoding a G protein-coupled receptorpolypeptide active in taste signaling having the nucleotide sequence ofSEQ ID NO:
 9. 135. An isolated nucleic acid molecule encoding a Gprotein-coupled receptor polypeptide active in taste signaling havingthe nucleotide sequence of SEQ ID NO:
 11. 136. An isolated nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:
 3. 137. Anisolated nucleic acid molecule having the nucleotide sequence of SEQ IDNO:
 13. 138. An isolated nucleic acid molecule having the nucleotidesequence of SEQ ID NO:
 15. 139. An isolated nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO:
 16. 140. An isolated nucleic acidmolecule encoding the polypeptide having the amino acid sequence of SEQID NO:
 4. 141. An isolated nucleic acid molecule encoding the Gprotein-coupled receptor polypeptide active in taste signaling havingthe amino acid sequence of SEQ ID NO:
 10. 142. An isolated nucleic acidmolecule encoding the G protein-coupled receptor polypeptide active intaste signaling having the amino acid sequence of SEQ ID NO:
 12. 143. Anisolated nucleic acid molecule encoding the polypeptide having the aminoacid sequence of SEQ ID NO:
 14. 144. An isolated nucleic acid moleculeencoding the polypeptide having the amino acid sequence of SEQ ID NO:17.
 145. An isolated nucleic acid molecule encoding a G protein-coupledreceptor polypeptide active in taste signaling comprising the consensussequence of SEQ ID NO: 18, or a consensus sequence having at least 75%identity to the sequence of SEQ ID NO:
 18. 146. An isolated nucleic acidencoding a G protein-coupled receptor polypeptide active in tastesignaling comprising the consensus sequence of SEQ ID NO: 19, or aconsensus sequence having at least 75% identity to the sequence of SEQID NO:
 19. 147. A genomic DNA amplified by a PCR reaction with at leastone degenerate primer having a nucleic acid sequence of SEQ ID NOs 5 or6, or consisting essentially of a nucleic acid sequence encoding aconsensus sequence of SEQ ID NO 18 or 19, wherein said amplified DNAcomprises a coding sequence for a G protein-coupled receptor polypeptideactive in taste signaling.
 148. A method for isolating a genomicsequence comprising a coding sequence for a G protein-coupled receptorpolypeptide active in taste signaling, said method comprising contactinga mammalian genome with at least one degenerate primer having a nucleicacid sequence of SEQ ID NOs 5 or 6, or consisting essentially of anucleic acid sequence encoding a consensus sequence of SEQ ID NO 18 or19, and amplifying said genomic sequence comprising said primer sequencein the presence of polymerase, free nucleotides and cofactors.
 149. Amethod for screening a mammalian genome for a coding sequence for a Gprotein-coupled receptor active in taste signaling, comprising: (i)contacting said mammalian genome with at least one degenerate primerhaving a nucleic acid sequence of SEQ ID NOs 5 or 6, or consistingessentially of a nucleic acid sequence encoding a consensus sequence ofSEQ ID NO 18 or 19; (ii) amplifying said genomic sequence comprisingsaid at least one primer sequence in the presence of polymerase, freenucleotides and cofactors; and (iii) detecting the presence of anamplified sequence comprising a G protein-coupled receptor polypeptidegene.
 150. Plasmid SAV115 comprising a mouse T1R3 gene.
 151. PlasmidSAV118 comprising a rat T1R3 gene.
 152. An isolated polypeptide selectedfrom the group consisting of: (i) a G protein-coupled receptorpolypeptide active in taste signaling encoded by a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs 9 and 11, and genomic sequences consisting essentially of SEQID NOs 1, and 2; (ii) a G protein-coupled receptor polypeptide encodedby a nucleic acid molecule comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs 3, 13, and 16, and a genomicsequence consisting essentially of a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs 15 and 20; (iii) a G protein-coupledreceptor polypeptide active in taste signaling comprising an amino acidsequence selected from the group consisting of SEQ ID NOs 10 and 12;(iv) a G protein-coupled receptor polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs 4, 14, and 17;(v) a G protein-coupled receptor polypeptide active in taste signalingencoded by a nucleic acid molecule comprising a nucleic acid sequencehaving at least about 50% identify to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs 9 and 11, and genomic sequencesconsisting essentially of SEQ ID NOs 1, and 2; (vi) a G protein-coupledreceptor polypeptide encoded by a nucleic acid molecule comprising anucleic acid sequence having at least about 50% identify to a nucleicacid sequence selected from the group consisting of SEQ ID NOs 3, 13,and 16, and a genomic sequence consisting essentially of a nucleic acidsequence selected from the group consisting of SEQ ID NOs 15 and 20;(vii) a G protein-coupled receptor polypeptide active in taste signalingcomprising an amino acid sequence that is at least about 40% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs 10 and 12; (viii) a G protein-coupled receptor polypeptidecomprising an amino acid sequence that is at least about 40% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs 4, 14, and 17; (ix) a variant of a G protein-coupled receptorpolypeptide active in taste signaling encoded by a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs 9 and 11, and genomic sequences consisting essentially of SEQID NOs 1, and 2, wherein said variant protein contains at least oneconservative substitution relative to the G protein-coupled receptorencoded by said nucleotide sequence; (x) a variant a G protein-coupledreceptor polypeptide encoded by a nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs3, 13, and 16, and a genomic sequence consisting essentially of anucleic acid sequence selected from the group consisting of SEQ ID NOs15 and 20, wherein said variant protein contains at least oneconservative substitution relative to the G protein-coupled receptorencoded by said nucleotide sequence; (xi) a variant of a Gprotein-coupled receptor polypeptide active in taste signalingcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs 10 and 12, containing at least one conservative substitution;and (xii) a variant of a G protein-coupled receptor polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs 4, 14, and 17, containing at least one conservativesubstitution.
 153. A fragment of the polypeptide of claim 152, whereinsaid fragment comprises at least about 5 to 7 amino acids.
 154. Thefragment of claim 153, wherein said fragment contains an extracellulardomain of a T1R mammalian G protein-coupled receptor polypeptide. 155.The fragment of claim 154, wherein said extracellular domain interactswith a compound involved in taste activation or modulation.
 156. Thefragment of claim 154, wherein said extracellular domain interacts witha protein involved in taste signal transduction.
 157. The fragment ofclaim 156, wherein said protein involved in taste signal transduction isa G protein subunit.
 158. The fragment of claim 157, wherein said Gprotein subunit is a promiscuous G protein.
 159. A chimeric or fusionpolypeptide comprising at least part of the amino acid sequence of apolypeptide of claim 152, and at least part of a heterologous amino acidsequence.
 160. The chimeric or fusion polypeptide of claim 159, whereinsaid heterologous sequence is a sequence from a different Gprotein-coupled receptor.
 161. The chimeric or fusion polypeptide ofclaim 159, wherein said heterologous sequence is a sequence from greenfluorescent protein.
 162. A method of screening one or more compoundsfor the presence of a compound that activates or modulates tastesignaling, comprising contacting said one or more compounds with one ormore fragments of one or more polypeptides according to claim 152,wherein the one or more fragments are at least about a 5 to 7 aminoacids in length.
 163. A method for screening one or more proteins forthe presence of a protein that interacts with a G protein-coupledreceptor active in taste signaling, comprising contacting said one ormore proteins with one or more fragments of one or more polypeptidesaccording to claim 152, wherein the one or more fragments are at leastabout a 5 to 7 amino acids in length.
 164. A polypeptide arraycomprising at least about a 5 to 7 amino acid segment of one or morepolypeptides according to claim 152, wherein said one or morepolypeptide segments are linked covalently or noncovalently to a solidphase support.
 165. An isolated antibody or antibody fragment that bindswith specificity to a polypeptide of claim
 152. 166. An isolatedpolypeptide having the amino acid sequence of SEQ ID NO:
 4. 167. Anisolated polypeptide having the amino acid sequence of SEQ ID NO: 10.168. An isolated polypeptide having the amino acid sequence of SEQ IDNO:
 12. 169. An isolated polypeptide having the amino acid sequence ofSEQ ID NO:
 14. 170. An isolated polypeptide having the amino acidsequence of SEQ ID NO:
 17. 171. A method for representing the perceptionof one or more tastes in one or more mammals, comprising the steps of:(i) providing values X₁ to X_(n) representative of the quantitativestimulation of each of n taste receptors of said mammals; and (ii)generating from said values a quantitative representation of tasteperception, wherein at least one of said taste receptors is a tastereceptor polypeptide having a sequence that is at least about 40%identical to a sequence selected from the group consisting of SEQ ID NOs4, 10, 12, 14, and
 17. 172. The method of claim 171, wherein saidrepresentation constitutes a point or a volume in n-dimensional space.173. The method of claim 171, wherein said representation constitutes agraph or a spectrum.
 174. The method of claim 171, wherein saidrepresentation constitutes a matrix of quantitative representations.175. The method of claim 171, wherein said providing step comprisescontacting a plurality of recombinantly produced taste receptors with atest composition and quantitatively measuring the interaction of saidcomposition with said receptors.
 176. A method for predicting the tasteperception in a mammal generated by one or more molecules orcombinations of molecules comprising the steps of: (i) providing valuesX₁ to X_(n) representative of the quantitative stimulation of each of ntaste receptors of said mammal, for one or more molecules orcombinations of molecules yielding known taste perception in a mammal,(ii) generating from said values a quantitative representation of tasteperception in a mammal for the one or more molecules or combinations ofmolecules yielding known taste perception in a mammal; (iii) providingvalues X₁ to X_(n) representative of the quantitative stimulation ofeach of n taste receptors of said mammal, for one or more molecules orcombinations of molecules yielding unknown taste perception in a mammal,(iv) generating from said values a quantitative representation of tasteperception in a mammal for the one or more molecules or combinations ofmolecules yielding unknown taste perception in a mammal; and (v)predicting the taste perception in a mammal generated by one or moremolecules or combinations of molecules yielding unknown taste perceptionin a mammal by comparing the quantitative representation of tasteperception in a mammal generated by one or more molecules orcombinations of molecules yielding unknown taste perception in a mammalto the quantitative representation of taste perception in a mammal forthe one or more molecules or combinations of molecules yielding knowntaste perception in a mammal, wherein at least one of said tastereceptors is a taste receptor polypeptide having a sequence that is atleast about 40% identical to a sequence selected from the groupconsisting of SEQ ID NOs 4, 10, 12, 14, and
 17. 177. A genomic DNAmolecule consisting essentially of a nucleic acid sequence coding for amammalian G protein-coupled receptor polypeptide active in tastesignaling comprising a consensus sequence selected from the groupconsisting of SEQ ID NOs 18 and 19, and sequences having at least about75% identity to SEQ ID NOs 18 or
 19. 178. An isolated RNA moleculetranscribed from the isolated DNA molecule of claim
 177. 179. Anisolated nucleic acid molecule that hybridizes to the DNA molecule ofclaim 177 under stringent hybridization conditions.
 180. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 177under moderate hybridization conditions.
 181. An isolated fragment ofthe genomic DNA molecule of claim 177 that is at least about 20 to 30nucleotide bases in length.
 182. A chimeric or fused nucleic acidmolecule, wherein said chimeric or fused nucleic acid molecule comprisesat least part of the coding sequence contained in the DNA molecule ofclaim 177, and at least part of a heterologous coding sequence, whereintranscription of said chimeric or fused nucleic acid molecule results ina single chimeric nucleic acid transcript.
 183. The chimeric or fusednucleic acid molecule of claim 182, wherein said heterologous codingsequence is from a sequence encoding a different G protein-coupledreceptor.
 184. The chimeric or fused nucleic acid molecule of claim 182,wherein said heterologous coding sequence is a sequence that facilitatesexpression of said mammalian G protein-coupled receptor polypeptide onthe surface of a cell.
 185. The chimeric or fused nucleic acid moleculeof claim 184, wherein said heterologous coding sequence is from amammalian rhodopsin gene.
 186. The chimeric or fused nucleic acidmolecule of claim 182, wherein said heterologous coding sequence is froma gene encoding green fluorescent protein or other detectable markergene.
 187. A cDNA sequence coding for a mammalian G protein-coupledreceptor polypeptide active in taste signaling comprising a consensussequence selected from the group consisting of SEQ ID NOs 18 and 19, andsequences having at least about 75% identity to SEQ ID NOs 18 or 19.188. An isolated RNA molecule transcribed from the isolated DNA moleculeof claim
 187. 189. An isolated nucleic acid molecule that hybridizes tothe DNA molecule of claim 187 under stringent hybridization conditions.190. An isolated nucleic acid molecule that hybridizes to the DNAmolecule of claim 187 under moderate hybridization conditions.
 191. Anisolated fragment of the genomic DNA molecule of claim 187 that is atleast about 20 to 30 nucleotide bases in length.
 192. A chimeric orfused nucleic acid molecule, wherein said chimeric or fused nucleic acidmolecule comprises at least part of the coding sequence contained in theDNA molecule of claim 187, and at least part of a heterologous codingsequence, wherein transcription of said chimeric or fused nucleic acidmolecule results in a single chimeric nucleic acid transcript.
 193. Thechimeric or fused nucleic acid molecule of claim 192, wherein saidheterologous coding sequence is from a sequence encoding a different Gprotein-coupled receptor.
 194. The chimeric or fused nucleic acidmolecule of claim 192, wherein said heterologous coding sequence is asequence that facilitates expression of said mammalian G protein-coupledreceptor polypeptide on the surface of a cell.
 195. The chimeric orfused nucleic acid molecule of claim 194, wherein said heterologouscoding sequence is from a mammalian rhodopsin gene.
 196. The chimeric orfused nucleic acid molecule of claim 192, wherein said heterologouscoding sequence is from a gene encoding green fluorescent protein orother detectable marker gene.
 197. A nucleic acid molecule comprisingthe isolated cDNA of claim 187 operably linked to a heterologouspromoter that is either regulatable or constitutive.
 198. The nucleicacid molecule of claim 197, wherein said regulatable promoter isinducible under specific environmental or developmental conditions. 199.A cDNA molecule comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs 3, 13, and
 16. 200. An isolated RNAmolecule transcribed from the isolated DNA molecule of claim
 199. 201.An isolated nucleic acid molecule that hybridizes to the DNA molecule ofclaim 199 under stringent hybridization conditions.
 202. An isolatednucleic acid molecule that hybridizes to the DNA molecule of claim 199under moderate hybridization conditions.
 203. An isolated fragment ofthe genomic DNA molecule of claim 199 that is at least about 20 to 30nucleotide bases in length.
 204. A chimeric or fused nucleic acidmolecule, wherein said chimeric or fused nucleic acid molecule comprisesat least part of the coding sequence contained in the DNA molecule ofclaim 199, and at least part of a heterologous coding sequence, whereintranscription of said chimeric or fused nucleic acid molecule results ina single chimeric nucleic acid transcript.
 205. The chimeric or fusednucleic acid molecule of claim 204, wherein said heterologous codingsequence is from a sequence encoding a different G protein-coupledreceptor.
 206. The chimeric or fused nucleic acid molecule of claim 204,wherein said heterologous coding sequence is a sequence that facilitatesexpression of said mammalian G protein-coupled receptor polypeptide onthe surface of a cell.
 207. The chimeric or fused nucleic acid moleculeof claim 206, wherein said heterologous coding sequence is from amammalian rhodopsin gene.
 208. The chimeric or fused nucleic acidmolecule of claim 204, wherein said heterologous coding sequence is froma gene encoding green fluorescent protein or other detectable markergene.
 209. A nucleic acid molecule comprising the isolated cDNA of claim199 operably linked to a heterologous promoter that is eitherregulatable or constitutive.
 210. The nucleic acid molecule of claim209, wherein said regulatable promoter is inducible under specificenvironmental or developmental conditions.
 211. A cDNA moleculecomprising a nucleic acid sequence having at least about 50% identity toa sequence selected from the group consisting of SEQ ID NOs 3, 13, and16.
 212. An isolated RNA molecule transcribed from the isolated DNAmolecule of claim
 211. 213. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 211 under stringenthybridization conditions.
 214. An isolated nucleic acid molecule thathybridizes to the DNA molecule of claim 211 under moderate hybridizationconditions.
 215. An isolated fragment of the genomic DNA molecule ofclaim 211 that is at least about 20 to 30 nucleotide bases in length.216. A chimeric or fused nucleic acid molecule, wherein said chimeric orfused nucleic acid molecule comprises at least part of the codingsequence contained in the DNA molecule of claim 211, and at least partof a heterologous coding sequence, wherein transcription of saidchimeric or fused nucleic acid molecule results in a single chimericnucleic acid transcript.
 217. The chimeric or fused nucleic acidmolecule of claim 216, wherein said heterologous coding sequence is froma sequence encoding a different G protein-coupled receptor.
 218. Thechimeric or fused nucleic acid molecule of claim 216, wherein saidheterologous coding sequence is a sequence that facilitates expressionof said mammalian G protein-coupled receptor polypeptide on the surfaceof a cell.
 219. The chimeric or fused nucleic acid molecule of claim218, wherein said heterologous coding sequence is from a mammalianrhodopsin gene.
 220. The chimeric or fused nucleic acid molecule ofclaim 216, wherein said heterologous coding sequence is from a geneencoding green fluorescent protein or other detectable marker gene. 221.A nucleic acid molecule comprising the isolated cDNA of claim 211operably linked to a heterologous promoter that is either regulatable orconstitutive.
 222. The nucleic acid molecule of claim 221, wherein saidregulatable promoter is inducible under specific environmental ordevelopmental conditions.
 223. The fragment of claim 153, wherein saidfragment includes at least an N-terminal fragment of a G protein-coupledreceptor.
 224. The fragment of claim 223, wherein said N-terminalfragment is involved in ligand binding.
 225. The polypeptide fragment ofclaim 224, wherein said fragment is at least about 100 amino acids inlength.
 226. The polypeptide fragment of claim 224, wherein saidfragment is at least about 600 amino acids in length.
 227. A biochemicalassay for identifying tastant ligands having binding specificity for a Gprotein-coupled receptor active in taste signaling, comprising: (i)contacting one or more fragments according to claim 224 with one or moreputative tastant ligands or a composition comprising one or moreputative tastant ligands; and (ii) detecting binding of a tastant ligandhaving binding specificity for said G protein-coupled receptor active intaste signaling.
 228. The assay of claim 227, wherein binding isdetected by displacement of a radiolabeled known binding ligand. 229.The assay of claim 228, wherein said known binding ligand is an antibodyor antibody fragment having binding specificity to said Gprotein-coupled receptor.
 230. An isolated nucleic acid molecule havingthe nucleic acid sequence of SEQ ID NO
 20. 231. A chimeric or fusionpolypeptide comprising at least an extracellular domain of at least onepolypeptide according to claim 152, and at least part of a heterologousamino acid sequence.
 232. The chimeric or fusion polypeptide of claim231, wherein said heterologous amino acid sequence is a sequence from adifferent G protein-coupled receptor.
 233. The chimeric or fusionpolypeptide of claim 232, wherein said different G protein-coupledreceptor is a T1R mammalian G protein-coupled receptor, and saidheterologous amino acid sequence includes at least an extracellulardomain of said T1R mammalian G protein-coupled receptor.
 234. Abiochemical assay for identifying tastant ligands having bindingspecificity for a G protein-coupled receptor active in taste signaling,comprising: (i) contacting one or more fragments according to claim 224with a preparation of G proteins and GTPγS, and one or more putativetastant ligands or a composition comprising one or more putative tastantligands; and (ii) detecting binding of a tastant ligand having bindingspecificity for said G protein-coupled receptor active in tastesignaling by measuring the binding of GTPγS to the G protein.